BI 1: BIN NP V NHN DNG CC TH BIN NP 1.

Nguyn tc - Bin np gene l qu trnh chuyn DNA (gene) vo t bo vi khun hay nm men nh mt vector (plasmid)_ y l k thut c s dng ph bin trong nhn dng phn t. - T bo kh bin l t bo trng thi c kh nng thu nhn (bin np) cc phn t ln nh DNA t ngoi mi trng vo trong t bo. - Thng thng cc t bo vi khun trong t nhin l cc t bo khng kh bin => x l bng cc tc nhn vt l (sc nhit, sc in) hay cc tc nhn ha hc (CaCl2) thu c cc t bo kh bin. - T bo mang plasmid l cc th bin np, n c pht hin nh cc gene cha trong plasmid (cc du chun chn lc). - Bi thc tp bin np gen s dng dng t bo E. coli DH5 v plasmid l pBluescript SK (l dn sut t plasmid pUC 19), mang gen chn lc l khng sinh ampicillin (amp1) v gen -galactosidase.

2. Nguyn liu, ha cht - Dung dch t bo vi khun E. coli DH5 kh bin - Dung dch dimethyl sulfoxide (DMSO) lnh - Dung dch plasmid - Mi trng Luria Bertani (LB) lng - Mi tng LB c thch - Dung dch CaCl2 0,1M - X-gal 20 mg/ml - IPTG 100Mm 3. Cc bc tin hnh

Ly 1 ng t bo kh bin t t -800C ra , lm tan , b sung 100ml dung dch CaCl2 0,1M c lm lnh trn , gi hn hp trn trong 10-15 pht - Thm vo dung dch t bo 3l DMSO cng c lm tan , gi tip trn - B sung 5ml dung dch plasmid (cha t 20-100 ng plasmid) trn u v tip trn 15 pht - Chuyn hn hp vo ni n nhit 420C trong 60 giy, sau t nhanh ln trong 2 pht - B sung 300 l mi trng LB lng gi 370C trong 30 pht phc hi sc sng ca t bo sau khi bin np c sc nhit. - Ht 30 l hn hp bin np vo ng eppendorf c cha 270 l mi trng LB lng ( pha long 10 ln d m v nhn dng cc khun lc sau khi cy) - Chun b 2 a thch c cha ampicillin ( ghi k hiu amp A1 v amp A10) v 2 a thch khng cha ampicillin ( k hiu 1X v 10X) - Cho vo mi a c cha ampicillin 20 l IPTG v 20 l X-gal, dng que gt gt u trn mt a thch - Ht 50 l dung dch t bo 1X cho vo 2 a thch tng ng, gt u. Tng t i vi dung dch t bo 10X - Chuyn cc t bo vo t m 370C nui cy qua m 4. Kt qu, gii thch - Quan st s hnh thnh cc khun lc trn a thch + Trn a thch khng cha ampicillin 1X ( hn hp bin np khng pha long) cc khun lc mc dy => t bo dng cho bin np c kh nng pht trin tt. Trn a thch 10X cc khun lc mc t hn v phn bit khi nhau. C th m c s lng cc khun lc trn a + Trn a thch cha ampicillin 1X cc khun lc mc rt nhiu , ch yu l khun lc mu trng, c s xut hin mt s khun lc mu xanh. Trn a thch 10X cc khun lc mc t hn rt nhiu, s lng cc khun lc mu xanh v trng u gim khong 10 ln so vi a thch 1X (do gene a vo c thit k vo vng m ha cho -galactosidase. Khi trong mi trng c X-gal ( 5-bromo-4-chloro-3-indolyl- -D-galactosidase), l c cht nhn to ca -galactosidase v cht cm ng isopropyl thiogalactopyranoside (IPTG) th t bo cha plasmid c mang gen mong mun cho khun lc mu trng, t bo cha plasmid khng mang gen s sn xut ra -galactosidase c kh nng phn hy X-gal thnh mt sn phm m sn phm ny khi c oxy s to thnh hp cht (5,5 dibromo4,4- dichloro indigo) c mu xanh da tri, nh vy s lm cho cc khun lc c mu xanh. ) + Tt c 4 a thch u khng b nhim nm, cc khun lc pht trin tt v s lng cc khun lc tng ng vi nng dung dch t bo nui cy => khng c sai st trong cc thao tc cng nh cc bc thc hin th nghim S lng khun lc mc trn mi a

+ Trn a khng cha ampicillin 10X c : 2000 khun lc + Trn a c cha ampicillin 10X c : 726 khun lc trng v 17 khun lc xanh T l khun lc xanh so vi s khun lc trng thp (2.3%) cho thy t l plasmid mang gene cn chuyn cao, tuy nhin, cng c th do tri IPTG v X-gal khng u. Hiu sut bin np l : H= Bin np tng i tt. = 0.3715

BI 2: TCH DNA PLASMID T T BO BIN NP 1. Nguyn tc - Da vo li th ca vic bin tnh bng kim i vi DNA plasmid v DNA nhn v s hi tnh mt cch c chn lc DNA plasmid sau khi trung ha dung dch. - DNA c tch chit c th c s dng trong cc phn ng ct bng enzyme gii hn, nhn dng v c trnh t. 2. Nguyn liu, ha cht - Dung dch I ( DD-I) ( glucose 50mM; m Tris- HCl, 25 mM, pH 8.0; EDTA 10 mM ) kh trng - DD-II (NaOH 0,2N; 1% SDS - DD-III (CH3COOK 5M: 60ml, CH3COOH: 11,5 ml, H2O: 28,5 ml, RNase 10mg/ml khng cha DNase - Phenol:chloroform:isoamylalcohol theo t l 25:24:1 v th tch - Cn tuyt i, cn 70% lm lnh -200C - m TE: 10mM Tris-HCl 10 mM, pH 8,0 c cha EDTA 1 mM - Isopropanol - Dung dch axetat kali 5M 3. Cc bc tin hnh - Ht 1,5ml dch nui cy t bo ( c chun b bng cch nui cy t 1 khun lc bi 1) cho vo ng eppendorf 1,5ml. ly tm 6000v/p ,4oC trong 3 pht. Loi b dch ni - Ha ta trong 100 l DD-I c lm lnh, vortex, 5 pht nhit phng - B sung 150 l DD-II, o ngc ng 5 ln , trn 5 pht - B sung 2000 l DD-III c lm lnh trn , o ngc ng 5 ln , trn 5 pht. Ly tm 14000v/p trong 10 pht. Thu 400 l dch ni sang ng eppendorf khc - Loi RNA bng cch b sung 2 l RNase , hn hp 37oC trong 20 pht - Cho 50 l hn hp Phenol:chloroform:isoamylalcohol, mix u. ly tm 14000v/p ,4oC trong 10 pht. Thu dch trong pha trn sang 1 ng eppendorf mi - B sung 300 l isopropanol, o ngc ng 5 ln , -20oC trong 5 pht. Loi b dch ni , p ng eppendorf ln giy thm ,ch mu kh - B sung 2 ln th tch cn tuyt i lm lnh -20oC. o ngc ng vi ln v gi -20oC trong 10 pht - Ly tm 14000 v/p ,4oC trong 10 pht ,nh nhng b dch ni. Ra ta bng cch b sung 1ml etanol 70% lnh, ly tm nh trn. Gn b dch ni , kh mu - Ha tan ta trong 30 l m TE. Mu thu c c th bo qun 4oC trong vi ngy hoc bo qun lu di -20oC. - o hp th bc sng 260 nm(A260) v 280 nm (A280) 4. Kt qu - Sau khi o ta thu c kt qu nh sau: A260 = 10.468 A260/A280 = 1.88

 DNA thu c c tinh sch cao, t ln protein v RNA Nng DNA c tnh theo cng thc: C (g/ml) = A260 x 50 x n Vi n l s ln pha long (n = 100) Nn ta c: C = 523.4 g/l

BI 3: K THUT PHN NG CHUI POLYMERASE (PCR) 1. Nguyn tc - Phn ng chui polymerase (PCR)l mt k thut cho nhn bn c hiu in vitro on DNA nm gia 2 vng trnh t bit, c tnh c hiu v nhy cao. Vic nhn bn DNA bng phn ng PCR i hi phi c cp mi c thit k da trn trnh t hai u ca on DNA cn nhn bn. - Thnh phn phn ng PCR: DNA khun, dNTPs, primers, DNA polymerase, m thch hp cho DNA polymerase. - Cc giai on chnh ca PCR: Giai on bin tnh: c thc hin nhit > 90oC, trong thi gian t 20 giy ti 1 pht. giai on ny DNA si kp s b bin tnh tch thnh 2 si n Giai on gn mi: thng thc hin 37-65oC ty thuc vo Tm ca mi. giai on ny cc mi bt cp vi 2 vng trnh t b sung ca DNA. Thng ko di 3060 giy. Giai on ko di: thc hin nhit ti u ca DNA polymerase vi s c mt ca dNTPs v di cc iu kin thch hp, kt qu l tng hp c mt si DNA mi b sung vi si DNA khun. Ty vo di on cn nhn bn m thi gian di ngn khc nhau. - Sau mi chu k ca PCR s bn sao ca on DNA cn nhn bn tng ln gp i, tuy nhin ch nn tin hnh 20-30 chu k do nng dNTPs trong mi trng gim,... 2. Nguyn liu, ha cht - Taq DNA polymerase - m ca Taq DNA polymerase (10X) - dNTPs nng 25mM cho mi loi dNTP - Mi T3 v T7 - DNA khun nng 10-25ng/l (thu c bi 2) - Dung dch nc kh trng - m TE 3. Cc bc tin hnh - Chun b mt ng eppendorf, cho vo : 58l nc 10l m 10l dNTPs Mi xui v mi ngc mi loi 8l Taq DNA polymerase 2l Trn u hn hp trn, sau chia u vo 4 ng eppendorf (k hiu t 1 ti 4) Ln lt thm vo cc ng t 1 ti 4 cc thnh phn theo th t sau: ng 1: 1l plasmid tch c (t bi 2)

ng 2: 1l plasmid ti t hp (i chng dng) ng 3: 1l nc(i chng m) ng 4: 1l plasmid nguyn bn => tng th tch mi ng eppendorf l 25l Trn nh, trnh to bt. Tin hnh PCR vi ch s nh sau: Bin tnh 94oC trong 1 pht Gn mi 55 oC trong 1 pht Ko di chui 72 oC trong 1 pht 30 giy Lp li 30 chu k t b sung bc gi 72 oC trong 7 pht cho qu trnh ko di hon tt. 4. Kt qu Thu c cc mu sn phm dng cho bi tip theo. -

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BI 4: PHN TCH DNA BNG IN DI TRN GEL AGAROSE Nguyn tc - Cc acid nucleic (DNA v RNA) c cha cc nhm phosphat trong phn t nn pH trung tnh chng l nhng polyanion => trong in trng chng s di chuyn t cc m v cc dng. - Cc phn t c kch thc khc nhau s di chuyn vi tc khc nhau trong cng mt in trng, trong phn t c kch thc nh s di chuyn nhanh hn v ngc li => c s phn tch thnh cc bng trn bn in di. - Gel agarose l mt cht nn c s dng cho in di, c kh nng phn tch cc on DNA c kch thc t 5kb ti vi chc kb ty thuc vo nng s dng - Sau in di, DNA c th c pht hin bng nhun gel vi ethidium bromide to phc cht c kh nng pht hunh quang di nh sng t ngoi,... Nguyn liu, ha cht - Bn gel agarose 2% - Thang chun DNA 1kb - Mu DNA cn phn tch Cc bc tin hnh - Trn m chm mu, trong m cn 1l v mu tng ng nh sau: Plasmid thu c sau PCR(sn phm tinh sch): 5 l Plasmid ti t hp(i chng dng): 7 l Plasmid nguyn bn: 7 l i chng m: 7 l Marker : 3 l - Cho mu vo cc ging tng ng trn bn in di. Ni my in di vi b ngun, ci t thng s (in di 110V, trong 30 pht). Kt qu Ta thu c bn in di nh sau:

Trong : Tnh t ging th 5 bn phi marker ta c:

Ging 5: plasmid thu c sau PCR Ging 6: plasmid ti t hp Ging 7: plasmid nguyn bn Ging 8 khng b sung g do mt mu i chng m. Quan st bn in di ta thy: - ging 5 v 6 c 2 bng nm gia 100bp v 200bp, bng ny chnh l on gene quan tm - ging 7 thy xut hin 2 bng. V ging 7 tra plasmid nguyn bn nn bng pha trn c th l cc plasmid dng gin xon, bng pha di l plasmid dng siu xon (do: dng siu xon c cu trc gn nn chy nhanh hn dng gin xon).