DNA extraction in Bacteria

( Escherichia coli).
GROUP 8
Calagui
Salaysay
So
Urbano
Yap
DNA
Deoxyribonucleic Acid

Organic chemical of complex molecular structure that is found in all

prokaryotic and eukaryotic cells and in many viruses.
DNA Extraction
process of isolating DNA from cells by mechanical and biochemical methods

Cells containing DNA will be opened chemically or mechanically to

release DNA

DNA is in a suspension, will be purified from contaminating cellular

components such as lipids in the cell membrane by washing it with SDS
or a detergent

DNA is precipitated using cold alcohol and resuspended in an

appropriate volume
DNA Extraction
Escherichia coli
bacterium that can be found in multiple places in the world but is most
commonly located within the intestines of human beings
Gram negative bacteria
Circular DNA
Methodology

1mL of overnight culture (Escherichia coli) was centrifuged at 13,000-16,000 xg for 2 minutes
Supernatant was removed and 600L of Nuclei lysis solution was added to the pellet
Solution was gently pipetted until cells were resuspended and was incubated at 80C for 5 minutes
Cell lysate was cooled to room temperature
3L of RNAse solution was added and the tube was inverted 2-5 times to mix the contents
Incubated at 37C for 15 minutes and was cooled to room temperature
200L of protein precipitation solution was introduced and the RNAse treated cell lysate was vortexed vigorously
at high speed for 30 seconds
The sample was incubated on ice for 5 minutes and centrifuged at 13,000-16,000 xg for 3 minutes
Methodology

Residue was discarded while the supernatant with DNA was transferred to a clean 1.5L microcentrifuge tube
containing 600L of room temperature isopropanol
Gently mixed by inversion until the threadlike strands of DNA form a visible mass
Centrifuged at 13,000-16,000 xg for 2 minutes and the supernatant was carefully poured off
Tube was drained on a clean absorbent paper
600L of room temperature 70% ethanol was added to the DNA before gently inverting the tube several times to
wash the DNA pellet
DNA was then centrifuged at 13,000-16,000 xg for 2 minutes, then ethanol was carefully aspirated afterwards
The tube was drained on a clean absorbent paper and the pellet was allowed to air dry for 10-15 minutes
100L of DNA rehydration solution was added to the DNA pellet before it was incubated at 65C for 1 hour
Solution was periodically mixed by gently tapping the tube and the resulting DNA was stored at 2-8C
Sodium dodecyl sulphate (SDS)
Detergent used to break down nuclear and cell membrane
Anionic detergent which emulsify lipid and protein components of the cell by disrupting
the noncovalent interactions that hold cell membrane together.

Ethylenediaminetetraaceticacid (EDTA)
Chelating agent that inactivates DNAse

An enzyme that breaks down DNA and

weakens the cell.
Tris-EDTA Buffer
dissolve / solubilize DNA while protecting it from degradation

NaCl
Sodium ions react with Phosphate PO4, forms Na+ shell, protects DNA from
destruction

Ethanol
DNA is insoluble in ethanol which causes its precipitation
UV Spectophotometry
Determines DNA concentration and purity
Absorbance at :
260 nm - absorbance peak of nucleic acids
280 nm- absorbance peak of pure proteins
Absorbance is correlated with amount of component
Absorbance Ratio
Defined by:
- A260= 1.0 pure DNA

Ratio of the absorbance of nucleic acids to the absorbance of proteins

A high absorbance ratio indicates a highly pure substance

1.8 commonly accepted value
Gel Electrophoresis
method for separation and analysis of macromolecules (DNA, RNA and proteins) and their
fragments, based on their size and charge.
Nucleic acid molecules are separated by applying an electric field to move the negatively charged
molecules through a matrix of agarose or other substances.
Possible sources of Error
not purposely denaturing the DNA when trying to remove DNase
incompletion of precipitating the DNA when adding the alcohol.
Contamination via not changing the pipette tips
lacked the appropriate heating measures by either too much or too little time
Conclusion
DNA extraction involves three main steps: lysis,purification, and precipitation.
DNA can be identified using different methods, like Gel Electrophoresis and UV
spectrophotometry.
References
Lzaro-Silva, D.N. & De Mattos (2015). J.C.P. The Use of DNA Extraction for Molecular Biology and
Biotechnology Training: A Practical and Alternative Approach. Journal of Creative Education, Vol 6, pp. 762-772.

Feng, Peter, et al. (2008). Enumeration of Escherichia coli and the Coliform Bacteria, in BACTERIOLOGICAL
ANALYTICAL MANUAL,8th Ed.

Skoog, Douglas A.; Holler, F. James; Crouch, Stanley R. (2007). Principles of Instrumental Analysis (6th ed.).
Belmont, CA: Thomson Brooks/Cole. pp. 169173.

Soovli, L.; Rm, E.-I.; Ktt, A.; et al. (2006). "Uncertainty sources in UV-Vis spectrophotometric
measurement". Accreditation and Quality Assurance. 11: 246255.