July 26 th August 6 th 2010 Course Run by Patricia Dyal Contact : +44 (0) 207 942 5059/5427 Email: pd@nhm.ac.uk Contents Page Course Timetable Introduction 1 Laboratory Health and Safety 2 Use of Pipettes 3 Section 1: Isolation of genomic DNA from Cestodes and Digeneans 4 1.1: Tissue solubilisation 5 1.2: Purification of DNA from cell lysate 6 1.3: Agarose gel analysis of purified gDNA 8 1.4: Nanodrop quantitation of DNA 10 Section 2: PCR amplification of a specific genomic region Large subunit 12 (LSU) and Small subunit (SSU) genes 2.1: PCR reaction setup using RTG beads 13 2.2: Agarose gel analysis of unpurified PCR reactions 15 2.3: Affinity column cleanup of PCR reactions 15 2.4: Agarose gel analysis of purified PCR reactions 17 2.5: Nanodrop quantitation of purified PCR product 17 Section 3: Cycle sequencing 18 3.1: Calculate the amount of DNA needed in sequencing reaction 18 3.2: Sequencing reaction setup 18 Section 4: Isolation of fish genomic DNA using Qiagen DNeasy DNA extraction 20 Kit 4.1: Tissue solubilisation 20 4.2: Purification of DNA from cell lysate 21 4.3: Agarose gel analysis of purified genomic DNA 23 4.4: Nanodrop quantitation of purified genomic DNA 23 4.5: PCR amplification of the mitoc hondrial cytochrome b (cytb) and 23 cytochrome oxidase gene (cox1) Section 5: CTAB genomicDNA Extraction and ethanol precipitation of 26 snail DNA 5.1: Tissue solubilisation 27 5.2: Organic extraction 28 5.3: Ethanol precipitation of genomic DNA 29 5.4: Agarose gel analysis of purified genomic DNA 31 Section 6: DNA Gel Extraction 32 6.1 QIAquick Gel Extraction Kit Protocol-using a microcentrifuge 32 6.2: Agarose gel analysis of purified PCR products 35 Section 7: Theoretical Background on Basic Molecular Techniques 36 7.1 DNA extraction 36 7.2 PCR Principles and Procedures 40 7.3 Agarose Electrophoresis 53 7.4 Purifiction of PCR products 57 7.5 Cycle Sequencing 61 7.6 Sequencher DNA sequencing assembly and analysis software 69 7.7 The basic local alignment search tool (BLAST) 73 Appendix 1: Buffers 74 Appendix 2: Tissue Storage 78 Appendix 3: IUB codes-Nomenclature for Incompletely Specified Bases in 81 Nucleic Acid Sequences Appendix 4: Consumables and Laboratory equipment 82 Outline Time Table: Week 1 26 th -30 th July 2010 1. General introduction Facility Health & Safety Training program 2. Set up DNA extractions overnight 3. DNA extractions 4. Gel electrophoresis of extracted DNA 5. PCR set up overnight-Kit based 6. Electrophoresis of PCR products 7. PCR products clean up for Sequencing 8. Quantification of PCR products 9. Set up sequencing reactions overnight. 10. Overview of Sequencing Facility with Julia Llewellyn Hughes (Sequencing facility Manager) 11. Overview of GENIUS bio-informatics software (Dr. Pete Olson) 12. Theoretical overview of all molecular techniques (principles and trouble shooting) Week 2- 2 nd -6 th August 2010 1. Meeting Dr. Phil Rainbow head of Zoology Department 2. Data Analysis Clean up of sequence data Types of Sequencing Software Blast searches Tree generating software Dr. Tim Littlewood (3 rd August) 3. DNA extraction using CTAB and recovery by organic extractions and ethanol precipitation. 4. PCR amplification of Mitochondrial genes using standard PCR reagents 5. DNA Gel extraction commercial kit based 6. Discussion: Basic laboratory equipment and consumables required for setting up a molecular facility. Storage of specimens for molecular analysis Storage of nucleic acids, short and long term 7. Meeting with Jackie Mackenzie Dodds (Laboratory Manager to discuss setting up a molecular collection. 1 Introduction The aim of this molecular training course is to provide initial experience of common laboratory and computing techniques used for generating and analysing molecular sequence data: Extraction and purification of genomic DNA using affinity spin column Use of agarose gel electophoresis to analyse DNA samples Amplification of a specific genomic target by the polymerase chain reaction (PCR) Purification of PCR products using affinity spin columns Automated DNA sequencing reactions DNA sequence editing DNA sequence identification and analysis using online and local software tools The theory and practice of phylogenetic reconstruction from aligned sequence data, and the different inference methods available In the laboratory practicals, you will be extracting DNA from cestodes (tapeworms) and digeneans (flukes). This will be a blind study, so from the DNA sequences generated you should be able to tell which specimens were cestodes or digeneans. If time permits we will also extract DNA from some fish specimens. 2 Laboratory Health and Safety Some of the reagents you will be using are toxic and PCR reactions are easily contaminated with external DNA from the environment, so it is important to wear lab coats and gloves whenever working at the bench to protect both you and your samples. Particular hazards will be detailed in the protocol. Waste solutions from the experimental process should be sealed in the reaction tubes and stored on your bench for collection and disposal no liquids from these protocols should be poured into the sinks unless specifically stated in the protocol. Empty tubes and discarded pipette tips should be placed in the clear plastic waste bags on your bench for disposal. Gloves, paper towels etc should be placed in the yellow sacs at the front of the laboratory. Wash your hands before leaving the laboratory. 3 Use of pipettors pipettors are delicate, expensive and liable to contamination never touch pipettors with bare hands never touch the barrel of the pipettor don't drop them onto the bench, place them gently maximum pipette volume is shown on the thumb knob, minimum volume is 10% of maximum never force pipettes beyond their stated volumes maximum volume minimum volume P2 2 l 0.2 l P20 20 l 2 l P100 100 l 10 l P200 200 l 20 l P1000 1000 l 100 l always set volumes by adjusting downwards from a larger volume (1/4 turn) older style models, with the small, round thumb knob, are adjusted using the internal collar, newer style models with the large, serrated thumb knob can be adjusted using either the thumb knob or the internal collar Pipettor settings P2 P20 P200 P1000 0 2 0 2 0 2 0 1 2 0 2 0 2 0 1 0 0 0 0 0 0 0 0 0 min max min max min max min max 0.2 l 2 l 2 l 20 l; 20 l 200 l 100 l 1000 l 4 Section1: I solationof CestodeAndDigeneangenomicDNA Background In order to isolate DNA from any specimen, you need to first release the DNA from the cellular compartments that it is held in (nucleus, chloroplast, mitochondrion etc) and then to separate it away from the other biomolecules present in that extract. There are a large number of protocols available to isolate either DNA or total nucleic acid, both "home-made" and commercial. Each technique has both advantages and disadvantages in terms of speed, complexity, use of toxic chemicals, cost and suitability to particular groups of organisms. Different types of cellular organisation (prokaryote vs. eukaryote, animal vs. plant etc.) can require different processing methods and special techniques may be required for particular taxonomic groups (eg. molluscs). You will be using a commercial protocol for the isolation of total nucleic acid (genomic and mitochondrial DNA plus RNA) from general animal tissues (Qiagens QIAamp DNA Mini Kit ). This method uses a pad of DNA-binding matrix supported on a mesh to trap the nucleic acids as a tissue lysate is spun through it in the presence of a salt buffer. The filter is then washed in ethanol-based wash buffers which remove any residual lysate whilst keeping the nucleic acid product bound to the matrix. The matrix is then dried and the purified, bound nucleic acid is eluted. The process involves 5 basic steps: Physical, chemical and enzymatic disruption of cellular structure Binding of DNA and RNA to a affinity column and removal of other cellular components by centrifugation Washing the bound nucleic acids by centrifugation Recovery of nucleic acids from the column by elution and centrifugation Examination of recovered DNA to assess yield, quality and purity 5 1.1 Tissue solublisation Tissue Samples: Cestodes And Digeneans 1. Otv 6. Nesc 2. Grill 7. Nesc 3. Gsq 8. Otb3 4. Gyrol 9. Elie 5. APHFP 10. Gono You are each supplied with: Reagent code Lysis buffer ATL Proteinase K (20mg / ml) PK Ultrapure dH 2 O in clear 7 ml bijou bottle H 2 O Cestode or Digenean in ethanol in clear 2 ml screw cap vial Unique ID 0.01TE pH 7.5 Consumables Sterile forceps 1.5 and 2.0 ml tubes Note, some of the buffers involved in this kit contain chaotrophic salts and must not be disposed of via the sink (contact with acids liberates cyanide!!!!!!) Therefore keep all liquid waste materials on the bench and they will be collected from you. Similarly, all tips and tubes used in this protocol must be placed in the plastic bags and will be collected for specialist disposal. 1. Make a note of your specimen ID code and label the 1.5 ml tubes with the specimen ID code. 2. Aliquot 500 l of 0.01 TE (10mM Tris-HCl pH7.5 / 0.1 mM EDTA) into a 1.5 ml tube. 6 3. Using the forceps remove sample tissue from ethanol and blot dry on a piece of clean tissue. 4. Cut off approximately 25 mg of tissue with a sterile scalpel blade and place the tissue in to the TE buffer. Return the remainder of tissue to ethanol. 5. Leave the tissue for a couple of minutes in the TE buffer to allow the ethanol to diffuse out. 6. Place the following into a 1.5 ml tube: 180 l of buffer ATL -tissue lysis buffer 20 l of Proteinase K solution (20 mg / ml stock) 7. Using the forceps, carefully remove the tissue and blot dry on a piece of clean tissue. Cut up the tissue into small pieces and transfer the to the lysis buffer. 8. Incubate tubes overnight in a the Thermomixer at 56C, mixing at 400rpm. 1.2 Purification of DNA from Cell Lysate Reagent code Binding buffer AL Ultrapure ethanol ETH Wash buffer 1 AW1 Wash buffer 2 AW2 Elution buffer AE Consumables QIAamp spin column 2 ml and 1.5 ml tubes Note, the buffers contain toxic chaotrophic salts and must not be disposed of via the sink. Keep all waste materials on the bench and they will be collected. 1. Collect your DNA extraction tube from the thermomixer. 2. Vortex the tubes for 15 seconds and then briefly centrifuge the tubes to remove drops from inside the lid. 7 3. Add 200 l of Binding Buffer AL to the tube and mix thoroughly by brief vortexing (15 seconds) 4. Incubate at 70C for 10 minutes. Briefly centrifuge the tubes to remove drops from inside the lid. 5. Add 200 l of ethanol (96-100%) to the tube and mix thoroughly by brief vortexing (15 seconds). Briefly centrifuge the tubes to remove drops from inside the lid. It is essential that the sample, Buffer AL, and the ethanol are mixed thoroughly to yield a homogeneous solution. A white precipitate may form on addition of ethanol. It is essential to apply all of the precipitate to the QIAamp Mini spin column. This precipitate does not interfere with the QIAamp procedure or with any subsequent application. 6. Unwrap the spin column / tube and pipette the mixture from step 5 into the top of the column 7. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute (ensure that the rotor is balanced before running the microfuge) 8. Transfer the column to a clean 2 ml collection tube 9. Add 500 l of Wash Buffer AW1 to the spin column 10. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute (ensure that the rotor is balanced before running the microfuge) 11. Transfer the column to a clean 2 ml tube and add 500 l of Wash Buffer AW2 to the spin column 12. Place the column / tube into a microfuge and spin at 14000 rpm for 3 minutes. 13. Transfer the column to a clean 2 ml tube and cut off the cap and hinge 14. Place the column / tube into a microfuge and spin at 14000 rpm for 1 minute . This dry spin ensures that the membrane is completely dry (elution of DNA from the membrane will fail if it retains any of the ethanol-containing wash buffer) 15. Transfer the column to a clean 2.0 ml tube and cut off the cap and hinge 16. Pipette 100 l of Elution buffer Buffer AE directly onto the membrane in the spin column (take care to not touch / puncture the membrane with the pipette tip) and leave for 5 minutes. 17. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute. 8 18. Leaving the spin column in the same tube, pipette a further 100 l of Elution buffer Buffer AE directly onto the membrane in the spin column (take care to not touch / puncture the membrane with the pipette tip) and leave for 5 minutes. 19. Place the column / tube into a microfuge and spin at 13000 rpm for 1 minute. 20. Remove and discard the spin column, and transfer the eluted, purified DNA (approximately 200 l) to a labelled, capped 1.5 ml tube. Note: Never vortex the eluted genomic DNA as this will fragment the high molecular weight DNA, just flick the tube to mix the DNA, spin briefly 1.3 Agarose Gel Analysis of Purified DNA Before you use a purified DNA extract, you need to confirm the success of the extraction (have you got any?), assess the quality of the extracted DNA (is it intact or broken up?) and determine its concentration (how much have I got?). This is initially done by running out a sample of your extracted DNA on an agarose gel, together with a reference DNA ladder containing DNA fragments of known size and quantity. Cross referencing of your sample to the bands of the ladder enables the size, quality and yield of your sample to be assessed. 1.3.1 Making the gel (for a 1% gel, 100mL volume) 1. Weigh out 1.0 of agarose into a 250 mL conical flask. Add 100ml of 1 x TAE, swirl to mix. It is god to use a large container, as long as it fits n the microwave, because the agarose boils over easily. 2. Microwave for about 3-4 minutes to dissolve the agarose. The agarose solution can boil over very easily so keep checking it. It is good to stop it after 45 seconds and give it a swirl. It can become superheated and NOT boil until you take it out whereupon it boils out all over you hands. So wear gloves and hold it at arms length. 3. Leave it to cool on the bench for 5 minutes down to about 60C (just too hot to keep holding in bare hands). If you had to boil it for a long time to dissolve the agarose 9 then you may have lost some water to water-vapour. You can weigh the flask before and after heating and add in a little distilled water to make up this lost volume. While the agarose is cooling, prepare the gel tank ready, on a level surface. 4. Add 10 l of Gel Red (5 l per 50 ml) and swirl to mix. Pour the gel slowly into the tank. Push any bubbles away to the side using a disposable tip. Insert the comb and double check that it is correctly positioned. 5. Leave to set for at least 30 minutes, preferably 1 hour. The gel may look set much sooner but running DNA into a gel too soon can give terrible-looking results with smeary diffuse bands. 6. Pour 1 x TAE buffer into the gel tank to submerge the gel to 25mm depth. This is the running buffer. 1.3.2 Preparing the samples 7. Into a clean 0.5 ml centrifuge tube place the following and mix by gentle aspiration: 3 l of your DNA sample 2 l of loading dye 8. Write in your lab-book the physical order of the tubes so you can identify the lanes on the gel photograph. 9. Load the first well with 5 l of the Hyperladder 1 size marker. Continue loading the samples and finish of with a final lane of marker. I load gels from right to left with the wells facing me. This is because gels are published, by convention, as if the wells were at the top and the DNA had run down the page. If this seems confusing then you can load left to right with the wells facing away from you. 10. Close the gel, tank switch on the power source and run the gel at 5V/cm. For example, if the electrodes are 10cm apart then run the gel at 50V. It is fine to run the gel slower than this but do not run any faster. Above 5V/cm the agarose may heat up and begin to melt with disastrous effects on your gel's resolution. Some people run the gel slowly at first (eg. 2V/cm for 10 minutes) to allow the DNA to move into the gel slowly and evenly, and then speed up the gel later. This may give better resolution. It is OK to run gels overnight at very low voltages, eg. 0.250.5V/cm. Check that a current is 10 flowing. You can check this on the power-source, the milliamps should be in the same ball-park as the voltage, but the best way is to look at the electrodes and check that they are evolving gas (ie. bubbles). If not then check the connections, that the power-source is plugged in etc. Monitor the progress of the gel by reference to the marker dye. Stop the gel when the bromophenol blue has run 3/4 the length of the gel. Switch off and unplug the gel tank and carry the gel (in its holder if possible) to the dark-room to look at on the UV light- box and photograph. Note: UV is carcinogenic and must not be allowed to shine on naked skin or eyes. So wear face protection, gloves and long sleeves. 1.4 Nanodrop Quantification of DNA Before proceeding further into costly and time-consuming manipulations it is critical to analyze, at least in a cursory way, the quantity and quality of DNA in the prep. A more accurate quantitation of your sample can be acheived using a "Nanodrop" UV spectrophotometer .The machine's use will be demonstrated to you. Only ever use non- scratching, non linting-tissues to clean the instrument. 1. Switch on machine 2. Start up the Nanodrop software and login. Select the "nucleic acid" option 3. Follow the onscreen instructions - place 1.5 l of ultrapure water onto the sample pedestal, close the arm and click on OK to initialise the instrument. 4. Once the instrument has initialised, lift the arm and carefully wipe the sample pedestal and arm Perform a blanking reaction using 1.5 l of whatever your sample is dissolved in (in our case, buffer AE), close down the arm and click on the blank button 5. Once the instrument has blanked (graph with zeroed baseline), lift the arm and carefully wipe the sample pedestal and arm 6. Perform a measurement - place 1.5 l of your sample onto the sample pedestal, name it in the sample ID window, close down the arm and click on the measure button. Note: It is advisable to blank the Nano drop after every sixth sample 11 7. Record the concentration (ng / l) and the OD 260/280 ratio: Estimating DNA Concentration by A260 The UV absorbance spectrum of DNA exhibits an Amax @ 260 nm based on the aromatic ring structures of the DNA bases. This is the most convenient way to estimate DNA concentration and calculate yield, as long as the DNA preparation is relatively free of contaminants that absorb in the UV. Proteins, and residual phenol left from the isolation procedure, are typical contaminants that may lead to an overestimate DNA concentration. An A260 = 1.0 indicates a [DNA] = 50 ug/ul, assuming the DNA is pure. Single-stranded DNA: 33 ug/ul RNA: 40 ug/ul 260/280: The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as pure for DNA; a ratio of ~2.0 is generally accepted as pure for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. 260/230 Ratio: This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for pure nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm 12 Section 2: PCR Amplification of A Specific Genomic Region Largesubunit (LSU) andSmall subunit (SSU) genes Background You will be amplifying a selected target region of the nuclear 18S (SSU) and 28S (LSU) gene for the cestodes and digeneans. Subsequently, you will use the PCR product as template in sequencing. For a PCR reaction, we need to combine, in the correct relative amounts: DNA template the 2 target-specific primer buffer dNTPs Taq polymerase To set up a PCR reaction it is possible to individually add each of these components. However, for simplicity, and standardisation, you will be using llustra PuReTaq Ready- To-Go PCR Beads Catlog no. 27-9559-01 (GE-Healthcare). These are commercial preparations of dNTPs, buffer and Taq polymerase, that have been mixed together, dispensed in the correct amounts for a single 25l PCR reaction and then lyophilised into a small bead that is stable for many months at room temperature. The additional advantage of using PCR beads is that they minimise the number of stock solutions and pipetting steps required for PCR reaction setup, thereby also minimising the risk of contaminating the reaction with DNA from external sources. This is especially important when using "universal" primers as they will also potentially amplify the gene from any contaminating DNA and PCR is a very sensitive process. Liquid PCR premixes are also available, some also include a gel loading dye so you can pipette straight from the finished PCR reaction onto a gel. 13 PCR and Sequencing Primers 2.1 PCR Reaction Set up using RTG Beads When setting up the reactions, use a new pipette tip for every pipetting step, pipette into the bottom of the tubes on the side without touching the bead and avoid touching the inside face of the cap. 1. Label the PCR bead tubes with sample ID and label one tube as the negative control tube. Write on the top of the side wall, not on the tube cap as this can get rubbed off. Always include a negative and a positive control when setting up your PCR. The negative control will give an indication of any reagent contamination. A positive control will confirm your PCR conditions are working. 2. To each label tube add the follwing: primer P1 10 M) 1 l primer P2 (10M) 1 l Sterile water 22 l DNA 1 l Note: If you are setting up several reactions using the same primer pair, it is best to prepare a master mix of the primers and water. This will reduce the number of pipetting SSU-18S LSU-28S PCR Primers Worm A + Worm B LSU-5F + 1500R Sequencing Primers: 300F ECD2S 930F 500F 1200F 900F 1270F 1200F 600R 300R 1200R 400R 1270R 1200R PCR product size 1700 bp 1500 bp 14 steps and the risk of contamination. For Example: I need to set up 10 reactions including a negative reagent control. X-1 X-10 primer P1 10 M) 1 l 10 l primer P2 (10M) 1 l 10 l DNA 1 l - Sterile water 22 l 220 l 25l 240 l Aliquot 24 l of the master mix to your label tubes, then add a 1 l of your respective template. 3. Vortex the tubes and spin briefly to bring the contents to the bottom of the tube. Once all the PCR reactions are set up, they will be cycled overnight under the following conditions: LSU -PCR Cycling Conditions Step Temperature (C) Duration Description # 1 95 5 min Initial denaturation x 1 2 95 30 sec Denaturation 3 55 30 sec Annealing x 40 4 72 2 min Extension 5 72 10 min Final extension x 1 6 4 hold x 1 SSU -PCR Cycling Conditions Step Temperature (C) Duration Description # 1 95 5 min Initial denaturation x 1 2 94 30 sec Denaturation 3 54 30 sec Annealing x 35 4 72 2 min Extension 5 72 10 min Final extension x 1 6 4 hold x 1 PCR reactions can be stored at -20 o C until purification. 15 2.2 Agarose Gel Analysis of Un-purified PCR Reactions 1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3.1 2. Into a clean 0.5 ml centrifuge tube, place the following and mix by gentle aspiration: 3 l of your PCR sample 2 l of loading dye 3. Carefully load your 5 l sample of (PCR reaction plus loading dye) onto the agarose gel (note the order in which you load your samples). 4. In a separate lane load 5 l of the molecular standard, Hyperladder 1, which allows you to determine the size of your PCR product. 5. The gel will be run at 60V for 45 minutes, examined and photographed under UV. 2.3 Affinity Column Cleanup of PCR reactions (Qiagen QIA quick PCR Purification Kit) Before the PCR product can be used in a DNA sequencing reaction, it must be purified from the other components of the PCR reaction mix (especially any unincorporated PCR primers, which would otherwise act as initiation points for additional, unwanted sequencing reactions and so give multiple, overlaying, sequencing reads from the same sequencing reaction). There are many techniques available to purify PCR products. Here we will be using a commercial affinity column method (Qiagen QIA quick Spin PCR cleanup columns). Like the QIAamp protocol, it uses a pad of DNA-binding matrix supported on a mesh to trap the PCR product as the PCR reaction is spun through it in the presence of a high salt buffer. The filter is then washed in an ethanol-based solution which removes any residual reaction components whilst keeping the PCR product bound to the matrix. The matrix is then dried and the purified, bound PCR product then eluted using a small volume of low salt buffer or water. However the column and buffers differ in formulation between the 2 kits. 16 Purify the amplified DNA from the remaining 22 l of your PCR reaction mix using the following protocol (note: do not purify the negative control which can now be discarded) Reagent code QiaQuick spin column in 2 ml centrifuge tube Buffer PB (DNA binding buffer) PB Buffer PE (wash buffer) PE Buffer EB (elution buffer, (10 mM TrisCl, pH 8.5)) EB Note, the buffers contain toxic chaotrophic salts and must not be disposed of via the sink. Keep all waste materials on the bench and they will be collected for specialist disposal. 1. To the remaining 22 l of PCR reaction add 110 l ( = 5 volumes) of Buffer PB and mix well by brief vortexing (2 seconds). 2. Apply the entire sample to the QIAquick column 3. Place the column / tube into a microfuge and spin at full speed (13000 rpm) for 1 minute (ensure that the rotor is balanced before running the microfuge) 4. Transfer the QIAquick column to a clean 2 ml tube and cut off the cap and hinge 5. Add 750 l of buffer PE to the QIAquick column 6. Place the column / tube into a microfuge and spin at full speed for 1 minute (ensure that the rotor is balanced before running the microfuge) 7. Transfer the column to a clean 2 ml tube and cut off the cap and hinge 8. Place the column / tube into a microfuge and spin at full speed for 1 minute (ensure that the rotor is balanced before running the microfuge) this dry spin ensures that the membrane is completely dry (elution of PCR product from the membrane will fail if it retains any of the ethanol-containing wash buffer) 9. Transfer the column to a clean 2 ml tube and cut off the cap and hinge 17 10. Pipette 30 l of Elution Buffer EB directly onto the membrane in the spin column (take care to not touch / puncture the membrane with the pipette tip) and leave for 1 minute Note: it is important that the elution buffer completely soaks into the membrane 11. Place the column / tube into a microfuge and spin at full speed for 1 minute (ensure that the rotor is balanced before running the microfuge) 12. Remove and discard the spin column, and transfer the eluted, purified PCR product (approximately 28 l) to a labelled, capped 1.5 ml tube 2.4 Agarose gel analysis of purified PCR Products 1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3 2. Into a clean, labelled 0.5 ml centrifuge tube, place the following and mix by gentle aspiration: 3 l of your purified PCR sample 21 l of loading dye 3. Carefully load your 5 l sample of (purified PCR product plus loading dye) onto the agarose gel (note the order in which you load your samples). 4. In a separate lane load 5 l of the molecular standard, Hyperladder 1, which allows you to determine the size of your PCR product. 5. The gel will be run at 60V for 45 minutes, examined and photographed under UV 6. After loading the gel, place the tube containing the remainder of your purified PCR reaction (approx 22 l) on ice. 2.5 Nanodrop quantitation of purified PCR product Accurately quantify your purified PCR product using the "Nanodrop" UV spectrophotometer as in section 1.4 18 Section3: CycleSequencing Background Sequencing both strands of the sequence of interest is highly recommended as it provides a double check for accuracy - data from each strand acts to verifying that from the other. Since (a) your PCR product is 1500 (28S) & 1700 base pairs long (depending on the source species) and (b) a single sequencing reaction is unlikely to read more than 800 bases at highest quality, we need to perform several sequencing reactions in both directions to obtain a the whole double stranded sequence. 3.1. Calculate the amount of DNA needed in sequencing reaction Need 2 ng of DNA for every 100bp of product e.g. Quantified 18S PCR product is 18 ng/l 18S PCR product is 700 bp long Therefore need 14 ng (2 x 7) of PCR product in the sequencing reaction # ng needed/ # ng per ul quantified = # l PCR product per 10 l seq rxn 14 ng / 18 ng / l = 0.78 l 3.2 Setting Up the Sequencing reaction 1. Remove the dilution buffer and big dye from the freezer and defrost on ice. 2. Mix the contents of each tube by vortexing and then spin tubes to remove contents from the lids. Keep tubes on ice . 3. Prepare your primer stock; this should be at 1 pmol/l. 4. Switch on the PCR machine to warm up. 5. Note: Your total sequence reaction volume is 10 l. Set up your sequencing reaction in 0.2 ml tubes as follows on ice: - DNA (PCR product) 0.78 l Primer (1 pmol/ul) 1.0 l Big dye dilution buffer from kit (2.5x) 2.0 l Sterile water 4.22 l Total 8.0 l 19 General note: If you are using the same primer for a number of templates you can prepare a master mix of primer and dilution buffer. e.g. 10 reactions using SSU 5F primer. Master mix:- SSU 5F 1 pmol/l 10 l Big dye dilution buffer (2.5x) 20 l Total 30 l Then aliquot 3.0 l of the master mix per tube. This saves time on pipetting and reduces the risk of cross contamination. 6. Mix the contents of your tube by gentle pipetting, if there are air bubbles or liquid on the side of the tube then give the tube a quick spin in the centrifuge. Return tube to ice. 8. Transfer tubes to PCR machine, select program call Big dye terminator. 9. Once the hot start section of the program is completed (5 min, pause 2 sec before this cycle is complete). 10. Transfer tube to ice, add 1 l of dilution buffer and 1.0 l of Big Dye terminator mix. Mix by gently pipetting up and down. Note: your total reaction volume is now 10 l. Note: If you are processing a large number of samples, you can prepare a master mix of the Big dye and dilution buffer. Aliquot 2.0 l of the master mix per tube, this increases pipetting accuracy and saves time on pipetting. 11. After the addition of the big dye return the tube to PCR machine and press resume, this will continue the program. PCR Profile: 96C 5 min Hot start 96C 10 sec 50C 05sec 28 cycles 60C 4 min The PCR should take about 2.30 hours to complete. 20 Section 4: I solation of fish genomic DNA usingQiagen DNeasy DNA extractionKit You will be using a commercial protocol for the isolation of total nucleic acid (genomic and mitochondrial DNA plus RNA) from general animal tissues (Qiagen DNeasy Blood & Tisue Kit). Tissue Samples: 1. LR3754 = Channa gachua 2. LR3731 = Luciocephalus pulcher 4.1 Tissue Solublisation You are each supplied with: Reagent code Lysis buffer ATL Proteinase K (20mg / ml) PK Ultrapure dH 2 O in clear 7 ml bijou bottle H 2 O Cestode or Digenean in ethanol in clear 2 ml screw cap vial Unique ID 0.01TE pH 7.5 Consumables Sterile forceps 1.5 and 2.0 ml tubes Note, some of the buffers involved in this kit contain chaotrophic salts and must not be disposed of via the sink (contact with acids liberates cyanide!!!!!!) Therefore keep all liquid waste materials on the bench and they will be collected from you. Similarly, all tips and tubes used in this protocol must be placed in the plastic bags and will be collected for specialist disposal. 9. Make a note of your specimen ID code and label the 1.5 ml tubes with the specimen ID code. 10. Aliquot 500 l of 0.01 TE (10mM Tris-HCl pH7.5 / 0.1 mM EDTA) into a 1.5 ml tube. 21 11. Using the forceps remove sample tissue from ethanol and blot dry on a piece of clean tissue. 12. Cut off approximately 25 mg of tissue with a sterile scalpel blade and place the tissue in to the TE buffer. Return the remainder of tissue to ethanol. 13. Leave the tissue for a couple of minutes in the TE buffer to allow the ethanol to diffuse out. 14. Place the following into a 1.5 ml tube: 180 l of buffer ATL -tissue lysis buffer 20 l of Proteinase K solution (20 mg / ml stock) 15. Using the forceps, carefully remove the tissue and blot dry on a piece of clean tissue. Cut up the tissue into small pieces and transfer the to the lysis buffer. 16. Incubate tubes overnight in a the Thermomixer at 56C, mixing at 400rpm. 4.2 Purification of DNA from Cell Lysate Reagent code Binding buffer AL Ultrapure ethanol ETH Wash buffer 1 AW1 Wash buffer 2 AW2 Elution buffer AE Consumables DNeasy spin column 2 ml and 1.5 ml tubes Note, the buffers contain toxic chaotrophic salts and must not be disposed of via the sink. Keep all waste materials on the bench and they will be collected. 21. Collect your DNA extraction tube from the thermomixer. 22. Vortex the tubes for 15 seconds and then briefly centrifuge the tubes to remove drops from inside the lid. 22 23. Add 200 l of Binding Buffer AL to the tube and mix thoroughly by brief vortexing (15 seconds) 24. Add 200 l of ethanol (96-100%) to the tube and mix thoroughly by brief vortexing (15 seconds). Briefly centrifuge the tubes to remove drops from inside the lid. It is essential that the sample, Buffer AL, and the ethanol are mixed thoroughly to yield a homogeneous solution. A white precipitate may form on addition of ethanol. It is essential to apply all of the precipitate to the DNeasy Mini spin column. This precipitate does not interfere with the DNeasy procedure or with any subsequent application. 25. Unwrap the spin column / tube and pipette the mixture from step 5 into the top of the column 26. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute (ensure that the rotor is balanced before running the microfuge) 27. Transfer the column to a clean 2 ml collection tube 28. Add 500 l of Wash Buffer AW1 to the spin column 29. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute (ensure that the rotor is balanced before running the microfuge) 30. Transfer the column to a clean 2 ml tube and add 500 l of Wash Buffer AW2 to the spin column 31. Place the column / tube into a microfuge and spin at 14000 rpm for 3 minutes. 32. Transfer the column to a clean 2 ml tube and cut off the cap and hinge 33. Place the column / tube into a microfuge and spin at 14000 rpm for 1 minute . This dry spin ensures that the membrane is completely dry (elution of DNA from the membrane will fail if it retains any of the ethanol-containing wash buffer) 34. Transfer the column to a clean 2.0 ml tube and cut off the cap and hinge 35. Pipette 100 l of Elution buffer Buffer AE directly onto the membrane in the spin column (take care to not touch / puncture the membrane with the pipette tip) and leave for 5 minutes. 36. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute. 37. Leaving the spin column in the same tube, pipette a further 100 l of Elution buffer Buffer AE directly onto the membrane in the spin column (take care to not touch / puncture the membrane with the pipette tip) and leave for 5 minutes. 23 38. Place the column / tube into a microfuge and spin at 13000 rpm for 1 minute. 39. Remove and discard the spin column, and transfer the eluted, purified DNA (approximately 200 l) to a labelled, capped 1.5 ml tube. 4.3 Agarose gel analysis of purified genomic DNA 1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3 2. Into a clean 0.5 ml centrifuge tube, place the following and mix by gentle aspiration: 3. 3 l of your genomic DNA 4. 2 l of loading dye 5. Carefully load your 5 l sample of (DNA plus loading dye) onto the agarose gel (note the order in which you load your samples). 6. In a separate lane load 5 l of the molecular standard, Hyperladder 1, which allows you to determine the size of your DNA. 7. The gel will be run at 60V for 45 minutes, examined and photographed under UV. 4.4 Nanodrop quantitation of purified genomic DNA Accurately quantify your purified genomic DNA using the "Nanodrop" UV spectrophotometer as in section 1.4 4.5 PCR amplification of the mitochondrial cytochrome b (cytb) and cytochrome oxidase (cox1) genes PCR and Sequencing Primers DNA Samples 1. LR3752 = Channa melasoma 2. LR3754 = Channa gachua cytb CO1 PCR Primers Part1-LRP211 + LRP21 Forward LRP140 + LRP142 PCR product size part 1 and 2 = 700 bp cox1 = 650 bp 24 3. LR3731 = Luciocephalus pulcher PCR Protocol 1. Defrost all PCR reagents, except the Taq Polymerase which is kept on ice. 2. Once thawed, vortex tubes and spin briefly. Place all tubes on ice. 3. Label the PCR tubes with sample ID and label one tube as the negative control tube. Write on the top of the side wall, not on the tube cap as this can get rubbed off. Always include a negative and a positive control when setting up your PCR. The negative control will give an indication of any reagent contamination. A positive control will confirm your PCR conditions are working. 4. To each label tube add the PCR components in the following order: Sterile water 13.8 ul 5 X Go Taq Flexi buffer 5 ul 25 mM Magnesium chloride 3 ul 10 uM dNTPs 1 ul Primer 1 0.5 ul Primer 2 0.5 ul Go Taq 5U/ul 0.2 ul DNA 1 ul 25 ul 5. Vortex the tubes and spin briefly to bring the contents to the bottom of the tube. Once all the PCR reactions are set up, they will be cycled overnight under the following conditions: PCR Cycling Conditions 25 Step Temperature (C) Duration Description # 1 94 3 min Initial denaturation x 1 2 94 1 min Denaturation 3 52-54 1 min Annealing x 35 4 72 1 min 30 sec Extension 5 72 5 min Final extension x 1 6 4 hold x 1 6. Run 3 ul of your PCR reaction on a 1% gel in TAE as in section 2.4. 26 Section 5: CTAB genomic DNA Extraction and ethanol precipitationof snail DNA Background Certain tissue types and organisms provide more of a challenge in terms of DNA extraction, with the "quick and easy" kit based techniques producing poor yields or damaged (sheared) DNA. Snails can be problematic, the actual tissue is tough and rubbery, so it is hard to physically break down and digest, and conventional techniques may shear the DNA. An alternative, "home brew" technique can produce better results. You will use a CTAB extraction technique coupled with organic extraction and ethanol precipitation to extract DNA from a specimen of Biomphalaria glabrata. CTAB (Cetyl Trimethyl Ammonium Bromide) is a powerful cationic surfactant which helps disrupt and lyse the snail tissue. Proteinase K is also used to break down proteins. Organic compounds such as phenol and "chloroform" (see below for explanation for the quote marks) are immiscible with aqueous solutions, yet denature and precipitate out proteins from them, whilst leaving nucleic acids untouched; different subsets of proteins are precipitated by the different organic compounds. Therefore mixing these compounds with the snail lysate and then separating the aqueous and organic phases by brief centrifugation (the aqueous phase is the top (lighter) one) clears the aqueous phase of proteins, which either form a pellet at the bottom of the tube or a layer at the interface between the phases. Because pure chloroform can be partly miscible with water, it is always used as a 24:1 mix with isoamyl alcohol, which allows for a clearer interface between the organic and aqueous phases. Hence "chloroform" refers to this 24:1 mix. Extraction can either be undertaken sequentially with phenol, then with a 1:1 mix of phenol-"chloroform", and finally with "chloroform", else 2 rounds of extraction with a 1:1 mix of phenol-"chloroform" can be performed In order to obtain DNA (actually total nucleic acids) from the resulting protein-free lysate, it s precipitated out of solution with alcohol and recovered by centrifugation. The DNA pellet is then washed, dried and redissolved in an appropriate aqueous buffer. Nucleic acids precipitate out of aqueous solution in the presence of >67% ethanol or >50% isopropanol. Either can be used, one the one hand, ethanol tends to 27 produce a slihghtly milky pellet whilst that with isopropanol is more glassy and so is harder to see, on the other hand, the total sample volume is larger with ethanol (volume of aqueous sample plus 2 volumes of 100% ethanol) than with isopropanol (volume of aqueous sample plus 1 volume of 100% isopropanol) and so, depending on sample volume, it may need to be divided between 2 tubes for the precipitation step (so mote handling time). Often salts are added to the aqueous phase to promote precipitation (eg ammonium acetate, sodium acetate, sodium chloride), hence the pellet-rinsing step, to eliminate salt carry over into the purified DNA solution. Note: both phenol and "chloroform" are toxic - standard laboratory schemes of work dictate that these steps are conducted in a fume hood. Because you will be using small volumes that have been pre-aliquoted for you, we have assessed that it is safe to conduct this practical exercise on the open bench. If you wish to use a fume cupboard, you are welcome to do so. All tips that are used during the organic extraction step must be kept separate for specialist disposal, and the organic phases must be sealed in tubes immediately after use, labelled and left on the bench for us to collect. 5.1 Tissue solublisation You are each supplied with: Reagent Code "x2" CTAB lysis buffer CTAB Proteinase K (20mg / ml) PK Frozen snail tissue Consumables Plastic petri dish Plastic microfuge pestle Screw cap 1.5 ml tube 1. Label the 1.5 ml screw cap tube. 28 2. Fresh or alcohol preserved animal tissue should be damp-dried quickly using a kimwipe, diced into small pieces using a scalpel blade. 3. Transfer the macerated tissue to the 1.5 ml screw cap tube and label it with your name 4. To the tube add 380 l of CTAB extraction buffer and 20 l of proteinase (20mg/ml stock solution). 5. Crush the tissue well with a microfuge pestle. 6. Incubate in the rotating incubator at 55C overnight 5.2 Organic Extraction You are each supplied with: Reagent Code Phenol:"chloroform" PC Chloroform:isoamylalcohol (24:1) Consumables 1.5 ml microfuge tubes 1. Transfer the snail lysate to a 1.5 ml microfuge tube 2. Place the tube into a microfuge and spin at full speed for 1 minute (ensure that the rotor is balanced before running the microfuge) to pellet residual undigested tissue. 3. Transfer the supernatant to a clean 1.5 ml tube, add 400 l of phenol / "chloroform" and firmly cap the tube 4. Gently invert the tube 20 times to form an emulsion. 5. Place the tube into a microfuge and spin at full speed for 3 minutes (ensure that the rotor is balanced before running the microfuge). 6. Transfer the aqueous (top) phase to a clean, labelled 1.5 ml tube. Try not to transfer any of the precipitated protein from the interface. It is better to leave a small amount of the aqueous phase behind. Cap the waste tube containing the organic phase. 29 7. Add 400 l chloroform / isoamyl alcohol (24:1) to the aqueous phase and firmly cap the tube 8. Gently invert the tube 20 times to form an emulsion. 9. Place the tube into a microfuge and spin at full speed for 3 minutes (ensure that the rotor is balanced before running the microfuge). 10. Transfer the aqueous (top) phase to a clean, labelled 1.5 ml tube. It is essential to not transfer any precipitated protein from the interface, or chloroform. Cap the waste tube containing the organic phase. 5.3 Ethanol Precipitation of genomic DNA You are each supplied with: Reagent Code 10M sodium acetate SA 100% ultrapure ethanol Eth 70% ultrapure ethanol 70 Ultrapure water H 2 O Consumables Disposable plastic pasteur pipettes Plastic petri dish 1.5 ml microfuge tubes 1. Add 40 l of 10M sodium acetate (one tenth of the volume) to the final aqueous phase from the organic extraction. 2. Add 880 l of 100% ethanol (ie. 2 x the combined volume of the original aqueous phase plus the acetate). 3. Vortex briefly to mix and then place in -70 o C freezer for 5 minutes. 4. Remove tube from freezer and allow to warm up on bench for a couple of minutes (if it goes straight into the centrifuge from the freezer, the tube will be brittle and may shatter). 30 5. Place the tube into a microfuge and spin at full speed for 5 minutes (ensure that the rotor is balanced before running the microfuge and that you place the tube into the centrifuge with its hinge pointing outwards). 6. Carefully remove the supernatant with a plastic pasteur pipette and discard into the petri dish (nb. The pellet may not be visible, but it will be on the side wall of the tube down from the cap hinge, slide the tip of the pipette slowly down the opposite wall of the tube, gently sucking up the liquid as you go). 7. Wash the pellet by adding 1000 l of 70% ethanol and vortexing briefly. 8. Re-pellet the DNA by spinning at 14000 rpm for 2.5 minutes (ensure that the rotor is balanced before running the microfuge and that you place the tube into the centrifuge with its hinge pointing outwards). 9. Carefully remove and discard the supernatant (as above). 10. Spin the tube again for a few seconds (ensure that the rotor is balanced before running the microfuge and that you place the tube into the centrifuge with its hinge pointing outwards) and remove the last of the liquid. 11. Place the tube, with its cap off, in the heat block at 37 o C for about 5 minutes to dry the pellet / evaporate any remaining drops of fluid (do not overdry the pellet - check it regularly and remove the tube as soon as there is no fluid left). 12. Once the pellet is dry, add 50 l of ultra water and incubate in the heat block at 37 o C for 30 minutes to redissolve the DNA. On 3- 4 ocassions during this period, gently tap the side of the tube to aggitate its contents. 13. Store extracted DNA at -20 o C Note: samples can be redissolved overnight at 4 o C as an alternative. Redissolving DNA in water, and storing at 4 o C is fine for short term storage (a few weeks maximum). For longer term storage, samples should be redissolved in TE buffer (10 mM Tris, 1 mM EDTA) and stored at -20 o C. For most purposes, repeated freeze-thawing will not harm the DNA. 31 Agarose gel analysis of purified gDNA You are each supplied with: Reagent code Loading dye LD Hyperladder 1 1. Prepare agarose gel as in section 1.3. 2. Into a clean 0.5 ml centrifuge tube place the following and mix by gentle aspiration: 3. 3 l of your DNA sample 4. 2 l of loading dye 5. Carefully load your 5 l sample of (DNA plus loading dye) onto the agarose gel (note the order in which you load your samples). 6. In a separate lane load 5 l of the molecular standard, Hyperladder 1, which allows you to determine the size of your DNA. 7. The gel will be run at 60V for 45 minutes, examined and photographed under UV. 32 Section6: DNA Gel Extraction DNA gel extraction is used to purify DNA from contaminants or to purify a specific fragment from a PCR reaction that has several fragments. 6.1 Agarose gel electrphoresis of PCR product 1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3. Noteusethewidecombswhenmakingtheagarosegel if thebandsaregoingto beexcised. 2. Into a clean 0.5 ml centrifuge tube, place the following and mix by gentle aspiration: 3. 22 l of your PCR sample (PCR sample remaining) 4. 5 l of loading dye 5. Carefully load your 27 l sample of (PCR reaction plus loading dye) onto the agarose gel (note the order in which you load your samples). 6. In a separate lane load 5 l of the molecular standard, Hyperladder 1, which allows you to determine the size of your PCR product. 7. Run the gel at 60V until it has run almost to the end, examined and proceed to section 6.2 below. 6.2QI Aquick Gel ExtractionKit Protocol-usingamicrocentrifuge This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per spin column. Notes: The yellow color of Buffer QG indicates a pH 7.5. Add ethanol (96100%) to Buffer PE before use (see bottle label for volume). Isopropanol (100%) and a heating block or water bath at 50C are required. All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge. 3 M sodium acetate, pH 5.0, may be necessary. 33 1. View the agarose gel on a longwave UV light box. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose. 2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 l). For example, add 300 l of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column. 3. Incubate at 50C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 23 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time. 4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 l of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.The adsorption of DNA to the QIAquick membrane is efficient only at pH 7.5. Buffer QG contains a pH indicator which is yellow at pH 7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding. 5. Add 1 gel volume of isopropanol to the sample and mix. For example, if the agarose gel slice is 100 mg, add 100 l isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage. 6. Place a QIAquick spin column in a provided 2 ml collection tube. 7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. The maximum volume of the column reservoir is 800 l. For sample volumes of more than 800 l, simply load and spin again. 8. Discard flow-through and place QIAquick column back in the same collection tube. Collection tubes are re-used to reduce plastic waste. 34 9. (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min. This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription or microinjection. 10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. Note: If the DNA will be used for salt sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 25 min after addition of Buffer PE, before centrifuging. 11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 13,000 rpm (~17,900 x g). I MPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. 12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube. 13. To elute DNA, add 50 l of Buffer EB (10 mM TrisCl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 l elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. I MPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 l from 50 l elution buffer volume, and 28 l from 30 l. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at 20C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM TrisCl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions 6.2 Agarose gel analysis of PCR product 35 You are each supplied with: Reagent code Loading dye LD Hyperladder 1 1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3 2. Into a clean 0.5 ml centrifuge tube, place the following and mix by gentle aspiration: 3. 3 l of your purified PCR product 4. 2 l of loading dye 5. Carefully load your 5 l sample of (PCR product plus loading dye) onto the agarose gel (note the order in which you load your samples). 6. In a separate lane load 5 l of the molecular standard, Hyperladder 1, which allows you to determine the size of your PCR product. 7. The gel will be run at 60V for 45 minutes, examined and photographed under UV. 36 Section 7: Theoretical Background on Basic Molecular Techniques 7.1DNA Extraction DNA Extraction or rather, nucleic extraction is the process by which nucleic acids are liberated from and then purified away from other cellular materials. There are three basic and followed by selective recovery of nucleic acids from the cellular lysate : 1. Physical Extraction- Breaking the cells open, commonly referred to as cell disruption or cell lysis to expose the DNA within. This is commonly achieved by grinding, homogenization, bead beating or sonicating the sample. 2. Chemical dissociation the solubilisation of lipids (cellular, nuclear, cytoplasmic and organellar membrane systems) using powerful detergent solutions (with appropriate buffer conditions (TRIS, EDTA)) such as: SDS (Sodium Docecyl Sulphate) CTAB (Cetyl Trimethyl Ammonium Bromide ) For bacterial plasmid preparation: SDS and NaOH is used to bust open the cells, followed by neutralisation and precipitation of cell wall and protein with potassium acetate mitochondrial DNA 3. Enzymatic dissociation DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol- chloroform and centrifuged the proteins will remain in the organic phase and can be drawn off carefully. The DNA will be found at the interface between the two phases (see below) 37 Physical Extraction: 1 by hand Motar and pestle Physical Extraction: 2 by machine Bead Beaters Rotating incubator 38 4. Recovery of Nucleic Acids 4.1 Organic Extractions to Precipitate Proteins phenol - (phenol + chloroform) chloroform Organic compounds such as phenol and chloroform are immiscible with aqueous solutions, yet denature and precipitate out proteins from them, whilst leaving nucleic acids untouched Different subsets of proteins are precipitated by the different organic compounds. Therefore mixing these compounds with a DNA lysate and then separating the aqueous and organic phases by brief centrifugation (the aqueous phase is the top (lighter) one) clears the aqueous phase of proteins, which either form a pellet at the bottom of the tube or a layer at the interface between the phases. Because pure chloroform can be partly miscible with water, it is always used as a 24:1 mix with isoamyl alcohol, which allows for a clearer interface between the organic and aqueous phases. Hence, chloroform refers to this 24:1 mix. Extraction can either be undertaken sequentially with phenol, then with a 1:1 mix of phenol-"chloroform", and finally with "chloroform", or 2 rounds of extraction with a 1:1 mix of phenol-"chloroform" can be performed 4.2 Alcohol Precipitation Recovery of nucleic acids by alcohol precipitation, usually ice-cold ethanol or isopropanol will aggregate together, giving a pellet upon centrifugation. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added. 39 4.3 By selective binding apply cell lysate to a physical matrix DNA binds to the matrix and is separated from other components of cell lysate on centrifugation. wash matrix elute nucleic acid from matrix to recover Silica based matrix 4.4 Recovery of nucleic acids: 3 considerations Taxon-specfic methods (CTAB) Environmental contamination: other organisms chemical contaminants eg. PCR inhibitors (humic acid in soil) Archival DNA from collections material Dried Fixed ethanol 40 formalin? 7.2 PCR PrinciplesandProcedures PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs(kb), although some techniques allow for amplification of fragments up to 40 kb in size. A basic PCR set up requires several components and reagents. [ These components include: DNA templatethat contains the DNA region (target) to be amplified. Two primers that are complemtary to the 3 (three prime) ends of each of the sense and antisense strand of the DNA target. Taqpolymerase with a temperature optimum at around 70 C. Deoxynucleotide triphosphates (dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions ; generally Mg 2+ is used, but Mn 2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn 2+ concentration increases the error rate during DNA synthesis [7] Monovalent cationpotassium ions. The PCR is commonly carried out in a reaction volume of 10200 l in small reaction tubes (0.20.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube 41 42 2.1PCR-PROCEDURE 43 I nitializationstep: This step consists of heating the reaction to a temperature of 9496 C (or 98 C if extremely thermostable polymerases are used), which is held for 19 minutes. It is only required for DNA polymerases that require heat activation by hot start PCR. Hot-Start PCR a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high- temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 9498 C for 2030 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA. Annealing step: The reaction temperature is lowered to 5065 C for 2040 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. Extension/elongationstep: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 7580 C, and commonly a temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'- phosphate group of the dNTPs with the 3-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used 44 and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential amplification of the specific DNA fragment. Final elongation: This single step is occasionally performed at a temperature of 70 74 C for 515 minutes after the last PCR cycle to ensure that any remaining single- stranded DNA is fully extended. Final hold: This step at 415 C for an indefinite time may be employed for short-term storage of the reaction. 2.2PCR Optimization 1. PCR Primer DesignGuidelines PCR involves the following three steps: denaturation, annealing and extension. First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice of temperatures is 94 o C, 60 o C and 70 o C respectively. Good primer design is essential for successful reactions. The important design considerations described below are a key to specific amplification with high yield. Primer Length- Typical primers are 18-28 nucleotides in length. A shorter primer such as a 15mer would have a higher chance of annealing at more than one complementary site within the genome. This may lead to amplification of nonspecific PCR products. Primer Sequence a) Avoid runs (3 or more) of Cs and Gs at the 3ends of primers as this may promote mispriming at G+C rich sequences. b) A thymidine (T) at the 3' end is not recommended, since it is more prone to mispriming than other nucleotides. 45 c) Avoid complementarity at the 3 ends of primer pairs as this promotes the formation of primer-dimer artefacts and reduces the yield of the desired product. (The creation and subsequent amplification of these primer dimers reduces the availability of primer to the template molecule resulting in decreased sensitivity or even failure of the PCR). d) Avoid sequences with significant secondary structure. Primer sequences should also be checked for self complementarity which could introduce secondary structures like hairpin loops into the primer. GC Content - The GC content (the number of G's and C's in the primer as a percentage of the total bases) of primer should be 40-60 % Primer Melting Temperature (T m )- by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58 o C generally produce the best results. Primers with melting temperatures above 65 o C have a tendency for secondary annealing. The GC content of the sequence gives a fair indication of the primer T m . Annealing temperature - The calculated Tm for a given primer pair should be similar. For this purpose, one can use the rule of thumb calculation of 2C for A or T and 4C for G or C (Thein and Wallace 1986). As a starting point, an annealing temperature 5C below the Tm can be used. This is usually the adjusted to improve specificity and yield in a series of optimization experiments. 2. Primer Annealing The temperature and length of time required for primer annealing depend on base composition, length and concentration of the amplification primers. An applicable annealing temp is 5C below the true Tm of the amplification primers. Increasing the annealing temp, increases specificity. It enhances discrimination against incorrectly annealed primers and reduces misextension of incorrect nucleotide s at the 3end of primers. Low extension temperatures together with high dNTP concentrations favours misextension of primers and extension of misincoporated nucleotides. 46 3. Primer Extension Extension time depends upon the length and concentration of the target sequence and upon temperature. Primer extensions traditionally performed at 72C, because this temperature was near optimal for extending primers on M13-based model template. Length of step is dependent on your target sequence length. In general, allow 1 min. per 1,000 bp of target to be amplified. An extension time of 5-10 min. at the end of the program allows for final extension of any unfinished products. 4. DenaturationTimeandTemperature The most likely cause for failure of a PCR is incomplete denaturaton of the target template and/or the PCR product. Incomplete denaturation allows the DNA tosnap back and, thus reduces the product yield. A high or too long denaturation step leads to unnecessary loss of enzyme activity. 5. DeoxynucleotideTriphosphates(dNTPs) Deoxynucleotide concentration between 20 and 200M each result in optimal balance among yield, specificity and fidelity. The four dNTPs should be used at equivalent concentrations to minimize misincoporation errors. Both the specificity and the fidelity of PCR are increased by using lower dNTP concentration. Lower dNTP concentration minimizes mispriming at non-target sites and reduces the likelihood of extending misincoporated nucleotides. 6. MagnesiumConcentration It is beneficial to optimize the magnesium ion concentration, as this is a necessary cofactor enzyme. A high magnesium concentration gives a higher yield, but also lower specificity. Magnesium ion concentration may affect all of the following:- 47 Primer annealing Strand dissociation temperatures of both template and PCR product Product specificity Formation of primer dimer artifacts Enzyme activity and fidelity Taq DNA polymerase requires free magnesium on top of that bound by template DNA, primers and dNTPs. PCRS should contain 0.5 to 2.5 mM magnesium over the total dNTP concentration. Note: the presence of EDTA or other chelators in the primer stocks or template DNA may disturb the apparent magnesium optimum. 7. EnzymeConcentration Recommended concentration range for Taq DNA polymerase is between 1 and 2.5 units. Requirements may vary with respect to individual target templates or primers. When optimizing a PCR, testing enzyme concentrations ranging from 0.5 units to 5units / 100l and assaying the results by gel electrophoresis. If the enzyme concentration is too high, non-specific background products may accumulate and if too low, an insufficient amount of desired product is made. 8. CycleNumber The optimum number of cycles will depend mainly upon the starting concentration of target DNA when other parameters are optimized. A common mistake is to execute too many cycles. You should not have to go over 40 cycles as a rule. Too many cycles can increase the amount and complexity of non-specific back ground products. Optimizing the number of PCR cycles is the best way to avoid to amplifying background products. Too few cycles give low product yield. 48 2.3PCR Troubleshooting 1. TheTemplateDNA The amount of total DNA in a PCR has a marked effect on the outcome of a PCR procedure. Using too much total DNA results in packed DNA in the confined space of the reaction vessel and can lead to false priming and even poor DNA synthesis due to the obstructed diffusion of large Taq polymerase molecules.The concentration of the target DNA should be balanced with the number of cycles in the reaction. Using an elevated concentration of the target combined with the normal, or higher than normal, number of cycles can cause the accelerated accumulation of nonspecific products. The accumulation of nonspecific products is often observed in a re-amplification PCR, when the high initial concentration of the PCR fragment is accompanied by a high number of cycles. Reducing the number of cycles may help. However, low concentrations of primer, target, Taq, and nucleotides are recommended as these generally ensure cleaner product and lower background. Impurities in nucleic acid preparations or in biological samples can inhibit or reduce the sensitivity and efficiency of PCR amplification 2. I nadequatedNTPs An incorrect concentration of deoxynucleotide triphosphates (dNTPs) can cause problems for the PCR procedure. The usual dNTP concentration is between 40M and 200M of EACH of the four dNTPs. Excessive dNTP concentrations can inhibit the PCR preventing the formation of product. Low primer, target, Taq and dNTP concentrations are preferable as these generally ensure cleaner product and lower background. 49 For longer PCR-fragments a higher deoxynucleotide triphosphate concentration may be required. A large change in the dNTP concentration may require a corresponding change in the concentration of MgCl 2 . Suboptimal concentration of nucleotides can cause incomplete primer elongation or premature termination of DNA synthesis during the elongation step of the PCR cycle. 3. Primer Concentration The concentration of primer in the amplification reaction should be between 0.1 and 0.5 M. For most PCR applications, including sensitive PCR assays and the amplification of longer PCR products, 0.2 M of each primer produces satisfactory results. High primer concentrations can have the following effects: Will promote mis-priming and accumulation of nonspecific product and may increase the probability of generating a template-dependent artifact termed a primer-dimer. If the primers are capable of forming dimers, raising their concentration only results in the creation of primer-dimers and does not improve the amplification of the desired PCR product. Non specific products and primerdimer artifacts generated are themselves substrates for PCR and compete with the desired product for Taq, dNTPs and primers, resulting in a lower yield of the desired product. Raising the primer concentration does not therefore cause an increase in the effective concentration of the primers. Low primer concentration generally ensures cleaner product and lower background. However, to amplify short PCR target sequences, careful calculation of the optimum primer concentration is required. For example, if the target fragment length is 100bp, a greater number of PCR product molecules is required to provide a specified amount of amplified DNA (in nanograms) than for a larger target fragment. In order to generate the required number of PCR product molecules, a greater number of primers 50 may be needed. Therefore, concentration of primers higher than 1M may be necessary, and desirable, for short target sequences. 4. TaqConcentration In a PCR experiment approximately 1 unit of the Taq enzyme should be used for a 25l reaction. Suboptimal concentration of the Taq enzyme can cause incomplete primer elongation or premature termination of the PCR product synthesis during the elongation step of a PCR cycle. Too much Taq will result in an excessive background of unwanted DNA fragments (a smear on a gel) while a huge excess may cause the reaction to fail with no product being detected. A Taq concentration of 1 unit per 25l reaction ensures a cleaner product and lower background. 5. MgConcentration Magnesium is a required cofactor for thermostable DNA polymerases. Mg 2+ in the PCR mixture stabilizes dsDNA and raises the Tm. Mg 2+ concentration therefore is an important for controlling the specificity of the reaction. A low Mg 2+ concentration requires more stringent base pairing in the annealing step. Too few Mg 2+ ions result in a low yield of PCR product. Too many Mg 2+ ions increase the yield of non-specific products and promote misincorporation as the fidelity of the Taq is reduced. On a gel this can appear as a ladder or smear. The MgCl 2 concentration should normally be between 1mM and 4mM Insufficient Mg 2+ concentration in a PCR mixture can causes failure of the reaction. The MgCl 2 concentration should normally be between 1mM and 4mM. Since dNTPs sequester Mg 2+ ions, a major change in the dNTP concentration in a rection would require a change in the concentration of MgCl 2 . Similarly, 51 changing the KCl-based buffer concentration or any other component of the PCR mix may require adjustment of the Mg 2+ concentration in the reaction mixture. 6. KCl Concentration Potassium chloride (KCl) is normally used in a PCR amplification at a final concentration of 50mM. To improve the PCR amplification of DNA fragments, especially fragments in the size range 100bp to 1000bp, a KCl concentration of between 70mM and 100mM is sometimes recommended. For the amplification of longer products a lower salt concentration appears to be better. PCR amplification of short products works better at higher salt concentrations. This is probably because an increase in salt concentration permits shorter DNA molecules to denature preferentially to longer DNA molecules. Shorter molecules are therefore amplified better at higher salt concentration. It should be remembered however that a salt concentration above 50mM can inhibit the Taq polymerase. An increase in KCl concentration may reduce the appearance of unwanted, long, non-specific products. Decreasing the KCl concentration to about 35 or 40mM, should get rid of short, non-specific products. In either case do not change the MgCl 2 concentration. To improve the yield of a product you can try adjusting the KCl concentration: increase it for a desired product less than 1000bp; lower it for a desired product greater than 1000bp. Consider thefollowingPCRmethods Nested PCR: A second PCR is performed on the product generated from the first PCR using primers, which are internal to the original primer pair. This improves sensitivity without impairing specificity. 52 Semi or hemi-nestedPCR: If a full nested PCR (using two internal primers) cannot be performed, sensitivity and specificity can be improved by using just one inner primer in conjunction with one of the outer primers from the first reaction. TouchdownPCR: Begin PCR cycle with a more stringent annealing temperature and decrease after each cycle until a predetermined annealing temperature is reached. This allows more stringent annealing of primers to template in the first few rounds of PCR, increasing the specificity of the PCR product. As specific products accumulate in the first rounds, they become available as templates in succeeding rounds which have decreased annealing temperature allowing for higher yield of product. 2.4Frequentlyasked Questions(FAQ) No Product Yield: 1. Is enzyme being added reproducibly? Consider making a master mix for multiple PCR reactions. 2. Are proper 0.2 ml tubes being used? The 0.2 ml tubes have thinner walls and allow for faster heat-transfer during temperature shifts. 3. Is denaturation step sufficient? Consider higher temperature or longer time. 4. Are primers stable? Reliable? Redesign or order new primers. Consider nested PCR (2 rounds of PCR with different primer sets). 5. Are there proteases or nucleases in sample? Consider a pre-incubation at 95C for 5-10 min before adding Taq. Use proteinase-K and inactivate at 95C for 5- 10 min before adding Taq. Low Product Yield - Optimize four key parameters: 1. Annealing temperature 2. Number of PCR cycles 3. Mg2+ concentration 4. Template concentration 53 Smear of PCR products on Gel How to get specificity: 1. Too much starting template? Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions. 2. Taq polymerase concentration too high? Use master mixes. 3. Mg 2+ concentration not optimal (too high)? Use buffer that allows separate addition of Mg2+. Perform a Mg2+ gradient from 0.55.0 mM (in 0.5 mM steps). 4. Are annealing or extension steps too long? Minimize times. 5. Primer concentration not optimal or primers degraded? Repeat the PCR with different primer concentrations from 0.10.5 M of each primer (in 0.1 M steps). 6. Too many PCR cycles? Reduce the number of cycles in steps of 3 cycles. 7. Annealing temperature too low? Optimize annealing temperature or redesign primers. Touchdown PCR (decrease annealing temperature each cycle). Hot-start PCR (withhold Taq until after initial 5 min. denaturation). 8. Primer design not optimal? Review your primer design, and design new primers. 9. Do primers have high complementarity to many sites on DNA? Blast primer sequences against template DNA sequence. Consider nested primer approach. Primer Dimer Formation 1. Are 3 ends of primer pairs complementary? Shift primer sequence up or down and reorder primers. 2. Are primers too short? Check primer characteristics. 3. Is template DNA concentration insufficient? Increase amount of template DNA added or re-test template DNA concentration. 4. Are primers too concentrated? Decrease primer amount or re-dilute primers from stock. 5. Too many cycles? Decrease number of cycles. 6. Annealing temperature too low? Increase annealing temperature or try gradient PCR. 54 7.3AgaroseGel Electrophoresis Agarose electrophoresis is a method used where charged molecules in solution, mainly nucleic acids and proteins, migrate in response to an electrical field. The rate of migration through the electrical field depends on: the net charge of the nucleic acid /protein the size and shape of the molecules the ionic strength, viscosity, and the medium through which the molecules are moving 3.1 Factorsaffectingmigrationof nucleicacids In an electric field, molecules migrate through the gel towards the anode (Negative-Positive) due to their overall negative charge. Nucleic acids have a natural negative charge due to their sugar-phosphate backbone. Separation of molecules on the gel is influenced by the molecule size and the effective pore size of the gel. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA moves. But voltage is limited by the fact that it heats and ultimately causes the gel to melt Conformations of DNA plasmid that has not been cut with a restricition enzyme will move with different speeds (slowest to fastest: nicked or open circular, linearised or suoercoiled plasmid) 3.2. Percent agaroseandresolutionlimits Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger molecules because they result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage agarose gels should be run with pulse field electrophoresis (PFE).The 55 following is a list of recommended gel percentages for the resolution of nucleic acids in electrophoresis: Gel percentage DNA size range bp 0.5% 1,000-30,000 0.7% 800-12,000 1.0% 500-10,000 1.2% 400 - 7,000 1.5% 200 - 3,000 2.0% 50 - 2,000 3.3 ElectrophoresisBuffers There are a number of buffers used for agarose electrophoresis. The most common being: Tris/Acetate/EDTA (TAE). TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. Tris/Borate/EDTA (TBE). TBE has a greater buffering capacity and will give sharper resolution than TAE. 3.4. Visualisationof DNA withGel Red Gel Red is used to make DNA or RNA bands visible in an agarose gel. It fluoresces under UV light (excitation at 300nm, emission at 595nm) when intercalated into DNA (or RNA) FEATURES Safer than EtBr (ethidium bromide) Easy disposal - Passed environmental safety tests for direct disposal down the drain or in regular trash Ultra-sensitive - Much more sensitive than EtBr and SYBR Safe Sensitivity: Bands of 0.25ng can be detected Extremely stable -Available in water, stable at room temperature for long-term storage and microwavable 56 Simple to use -Very simple procedures for either pre-cast and post gel staining Perfectly compatible with a standard UV transilluminator - Gel Red replaces EtBr with no optical setting change Dilution of Gel Red: 1. Add 2ul of the concentrated Gel Red stock to 1000 ul of Gel loading Buffer. Mix 2ul of this loading buffer with your sample. OR 2. Add 5 ul of Gel red to 50 ml of agarose solution. 3.5. Gel LoadingBuffer Loading dye is mixed with DNA samples for use in agarose gel electrophoresis. These buffers serve three purposes: They increase the density of the samples, ensuring that the DNA sinks evenly into the well. (glycerol, sucrose or Ficoll) They add colour to the sample, thereby simplifying the loading process. They contain dyes that in an electric field, move toward that anode at predictable rates. TrackingDyes The most common means of monitoring the progress of electrophoretic separations by following the migration of tracking dyes that are incorporated into the loading buffer. Two widely used dyes displaying different electrophoretic mobilities are bromophenol blue and xylene cyanol. Xylene cyanol typically migrates at approximately 4kb equivalence. So do not use this if you want fragments of 4kb. Bromophenol blue migrates at a rate equivalent to 200-400bp DNA. If you want to see fragments near this size (ie. Anything smaller than 600bp) then use the other dye because bromophenol blue will obscure the visibility of the small fragments. 57 3.6 Size Markers There are lots of different kinds of DNA size markers. In the old days the cheapest defined DNA was from bacteriophage so alot of markers are phage DNA cut with restriction enzymes. Many of these are still very popular eg, lambda HindIII, lambda PstI, PhiX174 HaeIII. These give bands with known sizes but the sizes are arbitrary. Choose a marker with good resolution for the fragment size you expect to see in you sample lanes. Companies have started producing ladder markers with bands at defined intervals, eg. 0.5, 1, 1.5, 2, 2.5kb and so on up to 10kb. If you know the total amount of DNA loaded into a marker lane, and you know the sizes of all the bands, you can calculate the amount of DNA in each band visible on the gel. This can be very useful for quantifying the amount of DNA in your sample bands by comparison with the marker bands. For example HyperLadder I is a typical ready-to-use molecular weight marker. Hyper Ladder I produces a pattern of 14 regularly spaced bands, ranging from 200 to 10,000bp. The 1,000 and 10,000bp bands have the highest intensity to allow easy identification. The size of each band is an exact multiple of 100bp. SIZING- when using the standard loading of 5l per lane, (720ng of DNA) each band corresponds to a precise quantity of DNA (see figure) 58 3.7 Troubleshooting Common problems encountered in agarose gel electrophoresis are described below, along with several possible causes. Poor resolutionof DNA fragments. The most frequent cause of poor DNA resolution is improper choice of agarose concentration. Low percentage agarose gels should be used to re-solve high-molecular-weight DNA fragments and high percentage gels for low-molecular- weight DNAs (see Table 2.5A.1). Fuzzy bands, encountered particularly with small DNA fragments, result from diffusion of the DNA through the gel. This is especially true when gels are run for long periods of time at low voltages. Bandsmearing. Trailing and smearing of DNA bands is most frequently observed with high molecular-weight DNA fragments. This is often caused by overloading the DNA sample or running gels at high voltages. DNA samples loaded into torn sample wells will also cause extensive smearing, as the DNA will tend to run in the interface between the agarose and the gel support. Meltingof thegel. Melting of an agarose gel during an electrophoretic separation is a sign that either the electrophoresis buffer has been omitted in the preparation of the gel or has become exhausted during the course of the run. For high-voltage electrophoresis over long time periods, TBE should be used instead of TAE as it has a greater buffering capacity. Also, minigel and midigel boxes, which typically have small buffer reservoirs, tend to exhaust buffers more readily than larger gel boxes. 7.4Purificationof PCR products Template purity and concentration are the two most important factors in obtaining good quality sequence data. Characteristics of poor quality templates: Noisy data or peaks under peaks No usable sequence data Weak signal 59 4.1ConsiderationswhencleaningPCR products Essential to separate the PCR product away from potentially interfering substances that may remain in solution after the PCR reaction. Primers, dNTPS, enzyme and salts should be removed. Determination of the presence of a single band or multiple bands that may result from your PCR reaction Primers Forward and reverse PCR primers remain in solution, will both act as sequencing primers. This results in multiple peaks from beginning to end as each primer can anneal to complementary strands with different nucleotide composition (multiple reactions) Results in overlapping fragments and unreadable data dNTPs Excess dNTPs will upset the specific ratios of dNTPs/ddNTPs required for optimal extension and termination in the cycle sequencing reaction. Salts The processivity of the Taq polymerase used in the cycle sequencing reaction declines in the presence of high amounts of salts Non Specific PCR products Primer dimer artifacts and secondary PCR products can result in poor quality sequence data. Nonspecific PCR products behave as templates in the sequencing reaction and produce extension products which results in noisy data 4.2Methodsfor purifyingPCR products 1) Ethanol precipitation 2) Column purification 3) Gel extraction (Qiagen kit) 1) Ethanol precipitation The PCR product is precipitated with ammonium acetate or sodium acetate and ethanol to remove the contaminants of the PCR Advantages Yield products of adequate purity for direct sequencing. 60 Disadvantages Ethanol contamination results in poor sequence data or complete failure, or Salt contamination in DNA preps results from co-precipitation of salts in alcohol precipitations insufficient removal of supernatant after precipitations or an incomplete wash of the pellet with 70% ethanol Salts can be preferentially injected and interfere with the migration of the DNA samples 2) Column purification methods I. Ultrafiltration Microcon Centrifugal Filter devices - Cost 2.00 per sample Millipore Montage PCR Clean up kit Automated 96 well format and half volume plate format; Cost-20 p per sample II. Affinity Column purification Qiaquick PCR purification columns (Qiagen); Cost - 1.38 per sample I. Ultrafiltration - Microcon Centrifugal filter device Microcon Spin columns have an ultrafiltration membrane across its bottom. The membrane has molecular size holes that allows molecules smaller than a predetermine size to pass through it under centrifugal force. Molecules larger than the membrane cut off size cannot pass through the membrane and are recovered from its surface. For example YM100 (100,000 dalton nonimal molecular weight) has a nucleotide cut of for double stranded DNA fragments of 125bp. The process of ultrafiltration uses semi-permeable membranes and pressure to separate molecular species on the basis of size and shape. Microcons, are disposable units that use centrifugal force to drive such separation. Microcon filters can be used to rapidly wash/desalt/buffer exchange, fractionate or concentrate many aqueous biological samples including nucleic acids. 61 Advantages Better alternative to ethanol precipitation. Reproducible and solute recoveries typically 95% of the sample Concentration factors as high as 100x II. Affinity Columns Qiagen QiAquick PCR Purification Columns Binds SS or DS DNA from 100bp-10 Kb Removes 99.5% of primers up to 40 nucleotides,dNTPs, salts Recovery of DNA 90-95 % QIAquick PCR Purification Procedure DNA absorbs to the silca membrane in the presence of high salt dNTPs, primer and salts pass through the column. Impurities are washed away Dry column Pure DNA is eluted with water 62 3) Gel Extraction This is the method of choice if your PCR reaction contains multiple bands. Run out all of the PCR on an agarose gel Excise out the correct size fragment and purify with Qiagen QIAquick (Gel extraction) spin columns Check purified DNA on a agarose gel Check the purity and concentration of the DNA using the Nano drop Calculate the amount of DNA required for your sequence reaction Obviously the deciding factor on which ultrafiltation method to use could be govern by the relative cost per sample. Millipore PCR purification kit - 20p Qiagen QIAquick PCR kit -1.38 Qiagen Gel extraction kit -1.38 Microcon filter units -2.00 7.5 CycleSequencing DNA sequencing is the process of determining the exact order of the bases A, T, C and G in a piece of DNA. In essence, the DNA is used as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA. Cycle sequencing is a modification of the traditional Sanger sequencing method. The components are DNA, primer, heat resistant DNA polymerase, 4 dNTPs, 4 ddNTPs (dideoxy terminator nucleotides) fluorescently labelled with four different dyes and buffer containing MG++ and K+. The single primer binds to the complementary DNA strand and is extended in a linear mode. This extension continues until by chance and depending on the complementary base a particular ddNTP is incorporated. Because of the latters dideoxy-configuration the polymerase cannot add any other base to this fragment and the extension is terminated. Thus at the end of the selected number of cycles, numerous fragments with different lengths and one labelled nucleotide at the end are 63 generated. Stoichiometric manipulation of the reaction components ensure that the fragments of every possible length starting from n+1 say 2000 bases are generated with n being the number of bases in the primer. The key difference between the traditional Sanger method and cycle sequencing is the employment of a thermo stable DNA polymerase. The advantage of using such a polymerase, is that the sequencing reaction can be repeated over and over again in the same tube by heating the mixture to denature the DNA and then allowing it to cool down to anneal the primers and polymerise new strands. Therefore less template DNA is needed than for conventional sequencing reactions. Also all 4 fluorescnce ddNTPS are in one single reaction, compared to 4 separate reactions for the old isotopic system. After a post sequencing reaction cleanup, the samples are electro kinetically injected into the array of 96-capillary sequencers. The negatively charged fragments migrate toward the anode by size, the smallest ones move fastest. Their tagged ddNTP terminators can be read as the fragments base sequence. A laser beam excites these dye molecules as the fragments reach a detection window, producing fluorescent signals that are collected from all 96-capillaries at once, spectrally separated and focused onto a CCD (charge coupled device) camera. Sophisticated optical and electronic devices produce a colour readout that is translated with the help of sequence analysis software into a sequence as we see it. template requirement (2-3ng / 100bp of PCR product) secondary structure Cycle sequencing reaction 64 T C G A Equivalence of manual and automated sequencing 65 O | H O O O H | | | | O = P - P - P - O - C - H | | | O O O | O | H base Deoxy-ribonucleotide RIBONUCLEOTIDE O | O | H O O O H | | | | O = P - P - P - O - C - H | | | O O O | O | H base Chain grows in this direction | O H | | O = P - O - C - H | O(-) O H | | O =P - O -C - H | O(-) O base | H | O | H 5 3 phosphodiester link O base | H | nextdNTP added here DNA replication O | H O O O H | | | | O = P - P - P - O - C - H | | | O O O | H base Di-deoxy-ribonucleotide O H | | O = P - O - C - H | O(-) O base | H | O | H | O O O H | | | | O = P - P - P - O - C - H | | | O O O O base | H | H O O O H | | | | O = P - P - P - O - C - H | | | O O O O base | H | H phosphodiester link chain grows no phosphodiester link chain is terminated 66 5.1PreparingtheSamples for CycleSequencing2-2AppliedBiosystems 3730/3730xl DNA AnalyzersSequencingChemistryGuide Template Types The following templates may be used with the BigDye terminator chemistry: PCR product Single-stranded DNA (e.g., M13) Double-stranded DNA Large DNA (e.g., BACs, PACs, YACs, cosmids, and fosmids) Bacterial genomic DNA Template Quality The quality of DNA in a reaction can affect the performance of the DNA Analyzer. When preparing DNA templates, it is critical to avoid the following: Residual salts Proteins Residual detergents Residual RNA The presence of residual salts, proteins, RNA, and detergents can interfere with capillary electrophoresis and electrokinetic injection. Your current template purification methods may have to be modified to remove residual salts, proteins, and detergents. The following are characteristics of poor quality templates: Noisy data or peaks under peaks No usable sequence data Weak signal Effect of Residual Salts Capillary electrophoresis is especially susceptible to salt in samples, either from template preparation, from cycle sequencing reactions, or from precipitation methods using salts. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to lower signal. In addition, the negative ions compete and interfere with the injection of larger DNA extension fragments, leading to shortened read lengths. 67 Effect of Proteins Many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary array, adversely affecting data resolution and capillary array lifetime. Effect of Residual Detergents Some methods of template preparation, such as the Thermomax method for M13 preparation, use detergents such as Triton X-100 to lyse the protein coat of phage particles. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will adversely affect the life of the capillary array and the quality of the sequencing data. Effect of Residual RNA Residual RNA that is present in DNA template preparations competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths. Template Quantity Effect of Too Little Template Too little template or primer in cycle sequencing reactions reduces the signal strength and therefore the peak height of reaction products. In the worst case, the signal-to noise level decreases so that bases cannot be called. Effect of Excess Template Excess template can affect data quality when: present in sample loaded onto the DNA Analyzer used in excess in the cycle sequencing reaction Excess template inhibits the injection of labeled extension fragments, thus affecting signals generated from this instrument. Excess template can behave similarly to 68 proteins and accumulate in the capillary array, which adversely affects data resolution and capillary array lifetime. Excess template used in the cycle sequencing reaction results in generation of short extension fragments. During electrokinetic injection, short fragments are injected more efficiently resulting in top heavy peak characteristics and shortened reads. (This phenomena becomes more pronounced with increased dilution of BigDye Terminator. Raw data from a BigDye terminator reaction containing excess DNA Template Below, analyzed data from a BigDye Terminator reaction contains excess template (this is from the same sample shown in above). The peaks are clearly off-scale and have overall shortened read lengths. The presence of excess template in the reaction and the preferential electrokinetic injection of small DNA fragments cause this effect QuickTimeand a TIFF (Uncompressed) decompressor are needed to see this picture. 69 QuickTimeand a TIFF (Uncompressed) decompressor are needed to see this picture. 70 7.6Sequencher DNA sequenceassemblyandanalysissoftware 1. Sequence Editing Sequencher gives you the DNA sequence editing tools you need to know that a sequence is absolutely correct. You can look at your chromatogram data one sequence at a time or view multiple aligned chromatograms in both forward and reverse orientations 2. Sequence Trimming Automated DNA sequencers occasionally produce poor quality reads, particularly near the sequencing primer site, and toward the end of longer sequence runs. The sequences of clones from DNA libraries frequently contain vector sequence, polyA tails, or other unrelated sequence. Introns and primer sequence frequently flank the sequence of QuickTimeand a decompressor are needed to see this picture. 71 amplified exons. Unless removed by trimming, any of these artifacts will distort your sequence assembly and downstream sequence analysis. Sequencher provides simple-to-use but powerful tools that help you trim poor quality or ambiguous data: Trim Ends removes misleading data from the ends of sequencing fragments. Trim Vector removes sequence-specific data contaminating the ends of your sequences. Trim to Reference eliminates the ends of sequences that extend beyond an assembled reference sequence. Sequence Assembly Sequencher's intuitive controls allow you to set your sequence assembly parameters and adjust them within seconds, allowing you to assemble your DNA fragments quickly and accurately. Sequencher will QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. 72 automatically compare the forward and the reverse-complement orientations to assemble the best possible contigs, so you can assemble DNA sequences regardless of orientation. Apply Sequencher's versatile assembly tools to: Compare gene variants to a reference sequence Confirm vector constructs Assemble viral and bacterial genomes Cluster tens of thousands of sequences from cDNA libraries Assemble cDNA to genomic sequence Create a primer map 4. Support for Confidence Values Sequencher displays confidence and summary confidence information (if available in your DNA sequence files) in the Project window, the Sequence Editor, and the Sequence Get Info window, so you can easily monitor the quality of your data. QuickTimeand a decompressor are needed to see this picture. 73 Genebank Feature Handling Sequencher imports GenBank Features in your files (if present) and has a variety of tools to help you manage, add, or edit Features. You can import Genbank Feature keys, or designate a key for your own features. The Define Feature Key Default Style user preference lets you set up your own colors and Feature annotations, so you could distinguish introns from exons, and variants from sequencing errors for example. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. 74 7.7 TheBasicLocal Alignment SearchTool (BLAST) The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. There are many different variations of BLAST available to use for different sequence comparisons, e.g., a DNA query to a DNA database, a protein query to a protein database, and a DNA query, translated in all six reading frames, to a protein sequence database. For a detail explanation of BLAST, please refer to the NCBI Handbook: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=handbook&part=ch16 75 APPENDI X 1- Buffers 1. MolesandMolar solutions(unit =M =moles/L) Sometimes it may be more efficient to use molarity when calculating concentrations. A mole is defined as one gram molecular weight of an element or compound, and comprised of exactly 6.023 x 10^23 atoms or molecules (this is called Avagadro's number). The mole is therefore a unit expressing the amount of a chemical. The mass (g) of one mole of an element is called its molecular weight (MW). The number of moles in an arbitrary mass of a dry reagent can be calculated as: # of moles = weight (g)/ molecular weight (g) Molarity is the unit used to describe the number of moles of a chemical or compounds in one liter (L) of solution and is thus a unit of concentration. By this definition, a 1.0 Molar (1.0 M) solution is equivalent to one formula weight (FW = g/mole) of a compound dissolved in 1 liter (1.0 L) of solvent (usually water). Example 1: To prepare a liter of a simple molar solution from a dry reagent Multiply the formula weight (or MW) by the desired molarity to determine how many grams of reagent to use: Chemical FW = 194.3 g/mole; to make 0.15 M solution use 194.3 g/mole * 0.15 moles/L = 29.145 g/L Example 2: To prepare a specific volume of a specific molar solution from a dry reagent A chemical has a FW of 180 g/mole and you need 25 ml (0.025 L) of 0.15 M (M = moles/L) solution. How many grams of the chemical must be dissolved in 25 ml water to make this solution? 76 #grams/desired volume (L) = desired molarity (mole/L) * FW (g/mole) by algrebraic rearrangement, #grams = desired volume (L) * desired molarity (mole/L) * FW (g/mole) #grams = 0.025 L * 0.15 mole/L * 180 g/mole after cancelling the units, #grams = 0.675 g So, you need 0.675 g/25 ml 2. Buffers 1. 50 x TAE (Tris-Acetate EDTA) Buffer pH 8.0 ( 1 litre) 242 g Tris base in 750 m ddH2O 57.1 ml Glacial acetic acid 100 ml 0.5M EDTA pH 8.0 Stir, do not adjust the pH Make up to 1000 ml Sterlize by autoclaving and store at room temperature. 1 x TAE Dilute to 1x concentration for the use in electrophoresis by taking 20ml of 50x buffer and making the volume to 1000ml by adding 980 ml of ddH2O. Final solute concentrations are 40 mM Tris acetate and 1 mM EDTA. The buffer is now ready for use in running an agarose gel. 2. 0.5M EDTA, pH8 Dissolve 186.1g EDTA (Ethylenediaminetetra-acetic acid disodium salt) in 800ml distilled water. Stir, on a magnetic stirrer with a 'flea' (magnetic stirring bar), and with the pH electrode in the buffer gradually add solid sodium hydroxide pellets (~20g of NaOH 77 pellets). Measure the pH and adjust until it is stable at pH8 with 5M NaOH solution. (As you add the NaOH the pH will come down. This allows more of the EDTA to dissolve and the pH will rise again - so you have to continue until the solution is clear and the pH is stable) Adjust volume to 1 liter with distilled water. Sterlize by autoclaving and store at room temperature. 3. TE (10mM Tris-HCL pH 7.5 / 0.1 mM EDTA pH 8.0) Stock Buffers required: 1 M Tris-HCL pH 7.5 and 0.5 M EDTA pH 8.0 For 50ml of TE buffer: VOLUME REQUIRED FOR 50 ML 1M Tris-HCL pH 7.5 O.5 ml (500 l) 0.5 M EDTA pH 8.0 0.01 ml (10 l) Sterile distilled water 49.49 ml 4. 2 x CTAB Extraction Buffer Weigh out chemicals with a face mask to avoid inhalation of dust. Tris (hydroxymethyl) amino methane 2.42 g Sodium Chloride 16.42 g Ethylenediaminetetra-acetic acid, disodium salt 1.2 g Hexadecyltrimethyl-ammonium bromide (CTAB) 4.0 g 2- Mercaptoethanol 0.4 ml For 200 mls of 2xCTAB: First dissolve the TRIS in 150 ml distilled water, then add EDTA, then the remaining chemicals and make up to 200 ml mark, using distilled water. Heating the solution gently may help to make the CTAB dissolve more quickly. 5. Phenol/ Chloroform / isoamyl alcohol (25:24:1) This can be bought commercially Prepare Phenol /chloroform/ isoamyl alcohol in 24: 1 ratio. For 50 ml mix the following: Phenol 25 ml Chloroform 24 ml Isoamylalcohol 1 ml Mix and store at 4C in a dark bottle. 78 6. Chloroform / isoamyl alcohol (24:1) This can be bought commercially Prepare chloroform/ isoamyl alcohol in 24: 1 ratio. For 50 ml mix the following: Chloroform 48 ml Isoamylalcohol 2 ml Mix and store at 4C in a dark bottle. General Note: The Molecular Cloning: A Laboratory Manual (Third Edition) (J oseph Sambrook, Peter MacCallum, David Russell) Volume 3 contain the recipes andmethodologytopreparestandardbuffersusedinmolecular biology. 79 Appendix 2: Tissue and DNA Storage I . TissueStorage 1. Cryo-preservation The ideal storage for molecular specimens is by using cryo-preservation, storage in liquid nitrogen or at -80C. 2. Storage in ethanol Ethanol preserved samples are regularly used in DNA molecular studies. However the quality of the alcohol used can play a large part in the condition of the DNA in a specimen. Preservation in absolute ethanol offers the best means of preserving both the DNA and the gross morphology of a specimen. DNA extracted from specimens preserve in absolute ethanol are of: High molecular weight High quality Disadvantages of ethanol preservation: Can cause extensive tissue shrinkage Colour loss Extract cellular components such as lipids 3. Storage in RNA later This reagent was designed for the storage of tissue samples from which you can extract RNA. However tissue stored in this buffer can be also used to extract DNA successfully of high molecular weight and quality RNA later is an aqueous, non toxic, tissue storage reagent that rapidly permeates most tissues to stabilize and protect RNA in fresh specimens. RNA later eliminates the need to immediately process or freeze samples; the specimen can simply be sub- merged in RNA later and stored for analysis at a later date. Storage and stability Store RNA later at room temperature. It is guaranteed for 6 months from the date of receipt, properly stored. Samples in RNA later can be stored for extended periods under conditions where RNA degradation would normally take place rapidly. Tissues can be stored indefinitely in RNA later at 20C or below. RNAlater can be safely discarded down the sink and flushed with water. Materials compatible with RNA later RNA later can be used for RNA preservation with most tissues, cultured cells, bacteria, 80 and yeast. RNA later may not be effective in tissues that are poorly penetrated by the solution, such as waxy plant tissue and bone. RNA later has been extensively tested with animal tissues including, brain, heart, kidney, spleen, liver, testis, skeletal muscle, fat, lung, and thymus. RNA later has also been proven effective for RNA preservation in E. coli, Drosophila, tissue culture cells, white blood cells, and some plant tissues. 4. Whatman FTA Cards FTA cards are designs for room temperature collection, shipment, archiving and purification of nucleic acids from a wide variety of biological samples for PCR analysis: Blood Cultured cells Buccal cells Plant material Bacteria Plasmids Microorganisms Solid tissue Viral particles FTA cards are impregnated with a patented solution that lyses cell membranes and denature proteins upon contact. Nucleic acids are immobilized and protected from UV damage and microbial fungal attack. FTA cards are available in several sizes to meet specific needs. Since captured nucleic acids are stabilized, FTA Cards facilitate sample collection in remote locations and simplify sample transport. For example, you can collect samples in the field without worrying about immediate refrigeration. Ship your samples back to the laboratory without expensive special handling or dry ice and process at your convenience. Store nucleic acids at room temperature for years Genomic DNA stored on FTA Cards at room temperature for over 17 years (and counting) has been successfully amplified by PCR. RNA, being chemically less stable than DNA, is best analyzed upon return of samples to the laboratory. Frozen storage is helpful for RNA preservation. Sample integrity is optimized when FTA Cards are stored in a Multi-Barrier Pouch with a Desiccant Packet. FTA Cards offer a compact room-temperature storage system that reduces the need for precious freezer space. I I . DNA Storage 81 Extracted DNA should be stored as follows: -20C or below for long term storage DNA for storage should be re-suspended in a buffer solution such as 10 mM Tris HCL pH 7.5 rather than water. If the pH of water is not neutral but slightly acidic this can cause the DNA to degrade upon storage. DNA can be stored lyophilized at -20C or below for long term storage 82 Appendix 3: I UB Codes-Nomenclaturefor IncompletelySpecified Basesin NucleicAcidSequences IUB Base Code Guide Standard Bases Mixed Bases (Wobble) Code Base B C, G or T D A, G or T H A, C or T V A, C or G R A or G Y C or T K G or T M A or C S G or C W A or T N Any base A, C, G or T Code Base A Adenosine C Cytidine G Guanosine T Thymidine 83 Appendix 3: Consumables and Laboratory equipment I -Consumables Description Supplier pack size KITS QIA quick PCR purification kit Qiagen 50 QIA quick PCR purification kit Qiagen 250 QIA quick gel extraction kit Qiagen 50 QIA amp DNA mini kit Qiagen 50 QIA amp DNA mini kit Qiagen 250 DNeasy blood and tissue kit Qiagen 50 DNeasy blood and tissue kit Qiagen 250 Taq DNA polymerase Qiagen 1000U DNEasy Plant mini kit Qiagen 50 Oligonucleotides oligo's unmodified ANY Taq GoTaq Flexi DNA polymerase Promega 100u - each dNTP dNTPs set 100 mM Bioline 4x25umol (4x250ul) Enzymes Proteinase K Sigma Genosys 500mg Gel Markers Gel marker - Hyperladder IV Bioline 200 Lanes Gel marker - Hyperladder I Bioline 200 Lanes GelRed gel stain Cambridge Bioscience 100ul Tips 0.1-10ul refill tips Starlab 10x96 1-200ul refill tips Starlab 10x96 200-1000ul refill tips (graduated) Starlab 10x96 0.1-10ul filter tips racked Starlab 10x96 0.5-10ul filter tips racked Starlab 10x96 1-20ul filter tips racked Starlab 10x96 20-200ul filter tips racked Starlab 10x96 200-1000ul filter tips racked Starlab 10x96 Fine tip mini pastettes Fisher 500 pack Tubes 0.2ml thin wall 8 tubes Strip Appleton Woods 120 Flat cap thin wall 8 cap strip Appleton Woods 120 0.2ml PCR Tubes, Flat Cap Fisher 1000 0.5ml microcentrifuge tubes Flat cap Starlab 5x200 1.5ml microcentrifuge tubes flat cap Starlab 500 pack 2ml microcentrifuge tubes flat cap Fisher 1000 pack 15ml centrifuge tubes -Falcon fisher 10 rack of 50 50ml centrifuge tube, non-skirted fisher 500 pack 7ml Aseptic, polypropylene, Bijou containers Fisher 1800 pack 30ml Disposable, Polystyrene, sterile universals Fisher 400 pack Gloves nitrile gloves small Semperguard Fisher 1000 pack nitrile gloves medium Semperguard Fisher 1000 pack 84 Description Supplier pack size nitrile gloves large Semperguard Fisher 1000 pack nitrile gloves xlarge Semperguard Fisher 1000 pack Miscellaneous Super Glossy Sony Thermal Paper UPP-110HA UVP 5 rolls in a box Sony Thermal Printer paper Sony UPP-110HA UVP 5 rolls in a box weighing boats anti static 30ml VWR 500 pack (80x60x14mm) weighing boats anti static 100ml Fisher 1x 250 pack dust masks (budget) autoclave tape - Steam ANY parafilm 100mm wide 38m long VWR 1 Roll (55mx19mm) saranwrap fisher 1 aluminium foil 75M x 450mm Western Laboratory 36 boxes Autoclave bag, polypropylene, without text, Chemicals VWR 500 pack (600x780mm) Agarose VWR 350 pack (700x1100mm) Ethanol absolute Disposable Pellet Pestles- Hand-operated or motor-driven grinders for resuspending pellets or disrupting soft tissue in microcentrifuge tubes. Pestle ends are specially designed to mate with 0.5 mL or 1.5 mL microtubes. Shaft is convenient length and diameter for gentle manual back-and-forth rotation or more vigorous motorized operation. QuickTimeand a decompressor are needed to see this picture. 85 II Laboratory equipment 1. ColdstorageFreezers& refrigerators 1. Ultracold -80 Freezer with a racking system 2. -20C freezer with a racking system 3. Refrigerator +4C 2. Autoclave 3. Ice Maker Machine Large volume refrigerated centrifuge-high speed 4. Laminar flow hood 5. Fume hood 6. Drying cabinet 7. Dish washer 8. Microwave 9. Magnetic stirrers with heating 10. Vortex mixer 11. Balances Fine balance (5 dp) Balance (3dp) or QuickTimeand a decompressor are needed to see this picture. 86 New Top of the range balance has dual mode from 5dp tp 3 dp depending on weight and anti static. Model: Qubis Sartorius Genuis balance MEM4145- supplied via VWR 12. Centrifuges: Microcentrifuges Bench centrifuges Refrigerated Benchtop centrifuge High Speed Centrifuge 13. Experion Bioanalyser-measures DNA/RNA/Proteins 14. Thermomixer Comfort- Supplier Eppendorf QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. 87 16. Nanodrop-spectrophotometer-Supplier Thermo Scientific ND1000-single channel ND8000-8 channel 17. PCR machines-Best to get gradient machines this is useful when optimising the annealing temperature for new primers. For example: Applied Biosystems Verity 96 Gstorm- GS00001 GS482 twin block GS482 twin block allows you to run to separate PCRs independently 18. Speedvac (vacuum concentrators)- to evaporate alcohol or dry down large volumes 19. Precellys 24-tissue homogeniser-Supplier Bertin Technologies QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. 88 20. UV transilluminator short wave 21. Long wave UV reader 21. UVP Gel documentation / digital camera system 22. Water baths 23. Dri heating block 24. Incubators Standard Air incubator Cool incubator Shaking incubator 25. pH meter 26. PCR cabinet- use to set up PCRs to reduce contamination QuickTimeand a decompressor are needed to see this picture. QuickTime and a decompressor are needed to see this picture. 89 27. Distilled water system 28. Horizontal electrophoresis unit with various comb sizes Mini Midi Maxi 29. Mini variable power supply for electrophoresis gel tanks 30. Adjustable Variable pipettes 1-10 l 2-20 l 10-100 l 50-200 l 100-1000 l 31. Freezer Storage boxes: Supplier Star lab (1) 1.5 and 2 ml tubes (PP) Catlog no: I2381-5040 StarStore 81-Place Storage Box (2) Storage box no dividers for 15 ml & 50 ml Falcon tubes Catlog no: E2300-5005 1. 2. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. 90 32. Tube Racks Racks for microcentrifuge tubes 0.2 ml, 0.5 ml, 1.5 ml, 2ml StarRack 96 96-place double-sided rack Transparent lift-off lid Holds 96 x 0.5ml tubes on one sideor 96 x 1.5/2.0ml tubes on the other Optional, 10-place acrylic rack available to help keep your racks tidy Temperature range: -90C to 121C Dimension (WxDxH): 222 x 114 x 28mm (without lid) / 50mm (with lid) PCR tube rack-0.2 ml Racks for 15 ml & 50 ml tubes 33. Ice Buckets QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. QuickTimeand a decompressor are needed to see this picture. 91 34. Lab Pal Label Printer The LAB PAL label printer is portable, easy-to-use and makes identifying your samples more quick and efficient. The long-lasting labels are designed to withstand extreme laboratory environments, such as liquid nitrogen, hot water baths, freezers and autoclaves. They also resist most chemicals, including Xylene, Alcohol, Ethanol and DMSO. 35. Howie Lab coats with elasticated cuffs. 36. Safety eye protection-goggles 37. Spatulas 38. Magnetic Stir bars 39. Robotic Station- Supplier Hamilton Robotics DNA extractions PCR set up PCR clean up etc. QuickTimeand a decompressor are needed to see this picture.