X L ETHIDIUM BROMIDE(EtBr) 1. S lc v EtBr: EtBr l cht c kh nng gy t bin, nu chm phi hoc ty ra khng ng cch s c nguy c b nhim EtBr.

y l cht dng trong cc phng th nghim sinh hc phn t, ch yu dng nhum (nh du) DNA/RNA khi chy in di. Hin nay c cc cch dng nh sau: + B sung trc tip EtBr vo dung dch m chy in di. + EtBr c b sung vo dung dch m load mu. + EtBr c b sung vo gel, lc cn cha c li, sau lc trn u, vi cch ny DNA (load mu) vn c nhum tt. + Mt vi qui trnh chy in di th thc hin nhum DNA sau khi chy in di. 2. Cc phng php loi b EtBr: Cc yu cu i vi cht thi EtBr: 2.1. Gel chy in di cha EtBr: Tt c nhng gel, cht rn c cha EtBr cn phi c thu nht v loi b khi mi trng lm vic nh cc cht thi c hi. Phi ng gi ring bit, ghi ch r l rc thi c cha EtBr. 2.2. Ethidium bromide solution: Nhng dch m cha nhiu hn 0,5mg/ml EtBr cn phi c lc, kh hoc thiu hy EtBr hoc lu tr li nh l nhng cht thi ha hc nguy him. Khi dung dch t hn 0,5mg/ml c th c thi b qua cng rnh. 2.3. Dng c thao tc i vi cc i tung c cha EtBr: Nhng dng c th nghim lin quan n EtBr cn nhng ni ring bit v lm du r rng.

3. Cc phng php x l EtBr hin nay trn th gii ang s dng: 3.1.Dng kit lc c bn sn loi b EtBr trong nc thi. Tc nhn lc ch yu l than hot tnh, nc thi sau khi lc c th ra cng rnh. Nhng b kit lc bn sn c cung cp t BIO 101, trong ch yu l than hot tnh ng di dng ti tr, mi ti c th hp th 10mg EtBr, mt b kit 50 ti nn c th hp th 500mg EtBr khi dung dch. Ti than sau khi x l cn loi b nh l cht thi ha hc c hi. 3.2. Loi b dung dch c nng thp (cha n 100mg/ml). 3.2.1. Cch 1 (Lunn v Sanrone 1987): + Thm 2,9g Amberlite XAD 16 cho mi 100ml dung dch x l. + Tr dung dch trong 12h nhit phng thnh thong lc u. + Lc dung dch qua giy lc Whatman No.1. b dch lc xung cng. + B giy lc v Amberlite vo ti nha, buc kn, loi b. 3.2.2. Cch 2 (Bensaude 1988). + Thm 300mg bt than hot tnh cho mi 100ml dung dch cn x l. + hn hp nhit phng, thnh thong lc u. + Lc hp hp qua giy lc Whatman No.1. b dch lc xung cng. + B giy lc v Amberlite vo ti nha, buc kn, loi b. 3.3.X l i vi nhng dung dch c nng m c (>= 0,5mg/ml) (Lunn v Sansone 1987). Bc 1: Thm nc h nng EtBr xung n 0,5mg/ml, hoc thp hn (thc hin trong t ht). Bc 2: B sung 20ml hypophosphorous acid 5% v 12ml 0,5mol Sodium nitrite, trn cn thn. (

Hypophosphorous thng c ly ra nng 50% sau pha long 10 ln c 5% ngay trc khi s dng).

Bc 3: Chun b sodium nitrite: Cn 3,45g sodium nitrite, sau ha tan vo nc, a v th tch 100ml Bc 4: Sau khi 24h nhit phng, a pH v trong khong 5-9 bng sodium bicarbonat. Sau b

(thc hin trong t ht). dch xung cng. * Khng s dng hypochloride x l EtBr. V x l vi thuc ty c th to sn phm gy t bin mnh hn v lng EtBr cn tha li sau khi x l l 20%. 3.4. Kh nhim b mt do EtBr. - Lau cht lng ri ra bng giy thm. B rc vo ti nilon, lm du buc kn v ni an ton. Nhng vt dng c nhim EtBr cn ring bit v nh du phn bit. CHIT SUT V TINH SCH AND I. GII THIU: Ton b ADN ca t bo, bao gm tt c gen v nhng vng lin gen c gi l b gen. B gen ngi cha khong 80.000 gen, nhng nhng vng m ha ca gen ny chim khong 3% ca ton b gen. B gen ca nm cha khong 6.000 gen. B gen ca mt vi thc vt c nhiu trnh t lp li. B gen ca prokaryote rt nh c cha trong nhim sc th c dng vng, prokaryote cng c th c nhng gen nm trong plasmid. B gen ca prokaryote khng c intron v nhng trnh t lp li. Mun nghin cu v b gen, k thut tinh chit ADN l mt trong nhng k thut sinh hc phn t u tin v cng quan trng gp phn cho s thnh cng ca nhng bc quan trng tip theo. Hin nay c nhiu phng php t phc tp, i hi nhiu thit b t tin, n cc phng php n gin hn, thi gian lm vic rt ngn ty theo mc ch s dng ADN, hay loi m, i tng no cn chit sut ADN. II. QUY TRNH CHIT XUT DNA CHUNG

1. Chun b mu: - i vi thc vt thng thu ly mu l, ng vt thu ly m trn c th, vi 2. Ph v t bo: - Dng phng php ly tm ph v mng t bo v phng thch ADN trong nhn. Ring mu l thc vt, do cn c lp v cellulose bn ngoi, nn phi c nghin trong nit lng, gip lm cho vch cellulose tr nn gin hn, d v hn, ng thi bo v ADN phng thch t nhn khng b tiu hy bi cc protease trong t bo. Ngoi ra, mng t bo v mng nhn cn c ph v bng hn hp cht ty (SDS sodium dodecyl sulfate, sarcosyl) v proteinase. Cc DNA s c gii phng ra mi trng. Cc protein lin kt vi DNA cng t b phn hy. 3. Ha tan ADN: - Ha tan DNA trong dung dch m buffer TE (pH 8) gip ADN khng b bin tnh. 4. Loi b cc thnh phn khng phi l ADN: - Proteinase K: loi b thnh phn protein. - CTAB: loi b polysaccharide v lipid. - Chloroform: phn tch v ko cc thnh phn khng phi l DNA xung mt di dung dch. 5. Ta ADN: - S dng ethanol 96% hoc isoproanol 200C ta DNA trong dung dch. Mc ch l thu nhn nucleic acid di dng c c v bo v chng khi s phn hy ca cc enzyme. 6. Ra DNA: - DNA c ra li bng ethanol 70% loi b cc mui v cc du vt isopropanol. 7. Lm kh: khun: nui vi khun trong mi trng nui cy t 15-16 gi tng sinh., thng nui trong 16 gi cho kt qu tt nht.

- DNA sau khi ra c lm kh bng cch phi nhit phng qua m hay sy kh. Mc ch trnh mu DNA cn cha ethanol s nh hng n DNA tng s. 6. Tr DNA: Tr ADN - 200C gip ADN khng b bin tnh. BI 1: QUI TRNH TRCH DNA T THC VT L chanh Von ka I. VT LIU- HA CHT 1. Vt liu sinh hc: L chanh Vonka. 2. Ha cht: Nit lng. Cn 70o EDTA. Agarose. Ethidium bromide. Isopropanol. SDS 10%. Chloroform. Isoamyl alcohol. Ethanol 96% v 70%. Nc ct.

Dung dch trch (Extraction buffer): 0.1M Tris.HCl (pH 8); 0.5M NaCl, 0.05M EDTA pH 8; 0.01% -mercaptho ethanol. CTAB buffer: 0.02M Tris.HCl pH 7.5; 2M NaCl, 0.05M EDTA. TE 0.1 buffer (10mM Tris pH 8, 0.1mM EDTA pH 8, chloroform/isoamylalcohol (24:1), ARNase (10 mg/ml). 3. Dng c Thit b Micropipet. My li tm. B in di minisub gel. Agarose. Tp Eppendorf 1,5 ml, 2,0 ml. Ko ct mu, chy v ci nghin mu. II. NGUYN TC T bo thc vt c nghin v trong iu kin lm lnh lm cho DNA c ha tan vo trong dung dch. SDS c thm vo gip DNA d ha tan hn. CTAB c s dng rng ri trch DNA cho hu ht cc loi ng vt, thc vt v vi sinh vt. CTAB lm cho DNA d ha tan, tch polisacharide v ARN ra khi DNA. Protein c tch khi DNA bng phenol hoc chloroform. Trong qu trnh trch DNA thc vt cn c gng trnh chn ng xoy lc mnh, nh th c th thu c DNA c chiu di t 50 100kb. III. PHNG PHP TIN HNH 1.Chun b mu: Mu l chanh Vonka c kh trng bng cn 70o (dng bng gn thm cn v lau u trn c hai b mt l) , dng ko ct ly phn tht l hai bn v b phn gn l gia. Phn tht l c gi trong giy nhm v ngm nit lng trong thi gian

10 pht. Sau , nghin k mu bng ci v chy c kh trng bng cch ngm nit lng. Trong qu trnh nghin ta thm nit lng vo nhm mc ch c ch cc enzym gy bin tnh DNA ca l, mu c nghin cho n khi thnh bt mn. 2.Trch DNA: Bc 1: ph v mng t bo v mng nhn: Chuyn phn bt mn vo tp 2,2ml. Sau cho vo tp 1ml dung dch EB c tc dng ph v mng t bo v mng nhn. T bo l c nghin vi dch trch EB (extraction buffer) ph v mng t bo, gii phng cc thnh phn c trong t bo ra ngoi mi trng dch trch. Tip tc cho 50ml dung dch SDS 10% vo tp c tc dng gip DNA d ha tan hn , sau lc nh tp(khng lc qu mnh DNA s b gy). Bc 2: mu: Mu sau khi thm cc dung dch EB v SDS 10% em nhit 65oC trong 30 pht, trong qu trnh khong 5 pht o nh tp trn mu. Bc 3: li tm: Sau thi gian , mu c em li tm vi vn tc 13000rpm trong 10 pht, tch cc thnh phn khc nhau ca t bo. Bc 4: ta DNA: Li tm xong ta ht 800ml phn dung dch trong pha trn cho vo tp mi, sau cho 800ml dung dch isopropanol vo tp v tin hnh trong thi gian khong 30 pht -20oC kt ta DNA, khng cho DNA bin tnh, gip tng kh nng thu DNA. iu kin ny nhiu protein v cc cht khc cng b kt ta cng ADN nn trong mu s b ln tp. Sau , tin hnh li tm vi vn tc 13000rpm trong 10 pht. Bc 5: ha tan ta: Li tm xong, phn dch trong c b vo mt ci hp m c cha nc v thu phn kt ta. Ha tan ta DNA trong 400ml TE, TE gi DNA n nh, khng b bin tnh. Bc 6: to phc vi CTAB: Sau khi ta ha tan ht ta cho thm vo tp 400ml CTAB v 65oC trong 15 pht, khong 5 pht o ngc ng tp trn u. Phng php ny s dng CTAB (Hexadecyltrimethylammoniumbromid) mt cht ty cation, ha tan mng t bo lm cho DNA d ha tan hn, bin tnh protein v hnh thnh phc hp vi nhng

polysaccharide, protein, polyphenol,. CTAB c kh nng lin kt thun nghch vi ADN v polysaccharide, c th nng mui cao (trn 1.4 M NaCl c trong m chit) th CTAB kt hp vi ADN v polysaccharide nhng phc hp CTAB-DNA tn ti trng thi ha tan, trong khi phc CTAB-polysaccharide li kt ta -phc hp ny sau c tch ra khi dung dch tch chit bng hn hp chloroform isoamyl alcohol. Bc 7: loi b protein v cc phc hp: Sau cho thm vo tp 800ml Chloroform/Isoamylalcohol lc u v li tm vi vn tc 13000rpm trong 5 pht loi b protein, nhng polycaccharide v phc hp trn. Li tm xong dung dch chia ra thnh 3 lp: lp th nht l phn dch trong trn cng c cha DNA, lp th hai l mt lp protein, lp k tip c cha chloroform v tt c nhng thnh phn cn loi b khc (polysaccharides, protein,). Bc 8: ta DNA: Sau khi ly tm ta tin hnh ht 500ml phn dch trong pha trn cho qua mt tp 2,2ml mi, thm vo tp ny 1ml Ethanol 96% nhit phng trong vng 15 pht ta DNA v bo v chng khi s phn hy ca cc enzyme. Sau , li tm 13000rpm trong 10 pht. Ethanol 96% c s dng kt hp vi mui trnh gy hi cho DNA. Bc 9: ra DNA: Li tm xong b phn dch trong pha trn vo hp m c cha nc, thu ly phn kt ta pha di. Sau , tin hnh ra kt ta vi ethanol 70% loi b mui ra khi DNA. Phn kt ta c ra 2 ln vi ethanol 70% (khng gy hi cho DNA do nng thp). Mi ln ra cho 400ml ethanol 70% vo tp, lc nh v em li tm trong 5 pht vi vn tc 13000rpm, sau loi b ethanol. Bc 10: phi kh DNA: Sau khi loi b ethanol, ta tin hnh phi kh DNA qua m nhit phng, trnh trng hp mu DNA cn cha ethanol s nh hng n DNA tng s, dn n nh hng n kt qu ca cc thao tc sau.. Bc 11: ha tan DNA: DNA sau khi phi kh, c ha tan trong 50ml nc. Sau , tin hnh pha long dung dch DNA trn: ht 90ml nc cho vo mt tp 1,5ml mi, sau ht 10ml dung dch DNA cho vo tp cha 90ml nc, lc nh. Tp 1,5ml ny c em i o OD. Dung dch DNA cn li trong tp 2,2ml l 40ml c dng chy in di.

3. Kim tra cht lng DNA: 3.1. Qu trnh chy in di: Phng php in di trong gel agarose l phng php thng dng dng tch ri cc on nucleic c kch thc t 200bp n 50kb. 3.1.1.Nguyn tc: - Nguyn tc ca phng php ny l da vo tnh phn cc ca phn t v kh nng di chuyn v mt hng xc nh khi chu tc ng ca mt in trng. Trong cc dung dch kim v trung tnh, cc i phn t nucleic acid tch in m ng u trn khp b mt nn trong in trng, chng s di chuyn v cc dng ca in trng. - Agarose l loi polyme tch t rong bin, c cu to bi 2 monome l t. - Cc on DNA kch thc khc nhau s chy vi tc khc nhau trn gel: on ngn chy xa, on di chy chm. ADN tng s c kch thc ln nht nn chy chm v chim v tr ln nht trn bn gel. 3.1.2.Quy trnh in di: + Chun b gel: Khay in di c t lc sn mt u khay. S dng lc c s rng l 17. Khay c t ni bng phng. Gel s dng chy in di l: 40ml vi nng 0,8% agrarose. Trc tin cn 0,32g agrarose cho vo chai thy tinh, sau cho vo chai 50ml Bufer TE, tr hao trong qu trnh nu bc hi ht 10ml. Lc chai cho agrarose ha tan vo Buffer TE, sau em nu trong microwave trong 2 pht 450W (nu cho agrarose ha tan hon ton v trong nh nuc), agrarose sau khi nu xong ngui khong 60oC ri nh dung dch vo D-galactose v 3,6-anhydro L-galactose, nn sau khi un si s to thnh mng li cho php cc phn t khc nhau i qua tu theo kch thc v trng lng phn

mt u khay dung dch s t tri u khay. Khong 45 pht sau gel c li, ta dng tay ly lc ra nhng phi cn thn trnh b b gel. t khay vo b in di, thm dung dch in di ph mt gel khong 2mm. Sau khi gel chun b xong, ta tin hnh load mu vo cc ging ca gel + Load mu: u tin ht dung dch loading buffer (mt dung dch ti DNA cha 30% glycerol, 0,25% bromophenol blue (BPB) v 0,25% xylencyanol (XC), trong thnh phn c cha glycerol c t trng cao s ko DNA nm trong ging khng b tri ra ngoi trong khi load mu) v nh thnh tng git nh khong 2ml ra giy paraflim, sau ht 10ml dung dch mu trn iu vi dung dch loading buffer v tin hnh load mu vo ging ca gel. Bt u load t ging th 2, ging th nht dng load thang chun. Sau khi load xong ta y np b in di li v tin hnh chy in di 25V. Thi gian in di thay i ph thuc vo ln ca DNA, nng agarose ca gel v cng dng in. Sau khi chy in di xong bn gel c ly nh ra khi khun gel v ngm vo dung dch EtBr trong thi gian 30 pht c tc dng nh du phn t DNA, ha cht ny c kh nng gn xen vo gia cc base ca nucleic acid v s pht hunh quang di tc dng ca tia t ngoi. Sau ra sch bn gel bng nc ct. Quan st v chp hnh: Bn gel c quan st di nh sng t ngoi Quan st thy ADN hin ln di dng cc vch sng. 3.1.3.Kt qu chy in di: 10 RNA Hnh 1. Hnh gel chy in di mu l chanh Vonka = 260-360 nm trn b c gel bio-rad.

Mu ging s 10, pha trn u ging c m trng chng t DNA cn nhim mui do mui cn st li trong qu trnh ra DNA bng cn ethanol 70%. Trong ging xut hin nhng vt sng chy di l do DNA b gy do trong qu trnh trch DNA ta lc mnh gn n gy DNA. m sng pha di cng l RNA do RNA c trng lng phn t nh hn DNA nn chy nhanh hn DNA. 3.2. Phng php o OD: 3.2.1. Nguyn tc: - Nguyn tc ca phng php ny l da vo s hp th mnh nh sng ca mt cht mt bc sng xc nh. Nucleic acid hp th mnh nh sng t ngoi bc sng 260 nm do s c mt ca base purine v pyrimidine. Gi tr mt quang bc sng 260nm (OD260nm) ca cc mu cho php xc nh nng nucleic acid trong mu. . n v OD260nm tng ng nng 50ng/ml cho mt dung dch ADN si i. Do nng ADN trong mu c tnh theo cng thc sau: CADN (ng/ml) = OD260nm * 50 * pha long - kim tra sch ca dung dch, ngi ta o thm gi tr OD 280 nm (OD280nm). bc sng ny, cc protein c mc hp th cao nht. Ngoi ra, cc protein cng hp th nh sng bc sng 260 nm nh cc nucleic acid v do lm sai lch gi tr tht ca nng nucleic acid. Mt dung dch nucleic acid c xem l sch (khng tp nhim protein) khi t s : OD260nm/OD280nm nm trong khong 1.8 2 3.2.2 Cc bc o OD: - Ly 100ml nc kh ion v trng vo cuvet 100ml, o i chng. - Ho 10ml dch ADN cn o vo 90ml nc kh ion v trng, bm vo cuvet, o bc sng 260nm, 280nm - c hp th trn my o quang ph bc sng 260 nm, 280 nm. X l s liu theo cng thc trn.

3.2.3. Kt qu o OD: Sau y l bng th hin kt qu o OD ca mu l chanh Vonka: 260nm 3,0291 280nm 2,9259 260/280 1,0353 280/260 0,9659

Ta c: OD260nm = 1 DNA = 50ng/ml Qua kt qu o OD ca l chanh Vonka ta c: OD260nm = 3,0291 Trc khi tin hnh o OD, mu c pha long 10 ln. Vy nng DNA trong mu l chanh Vonka thu c: CDNA= OD260nm * 50ng/ml*10 = 3,0291*50ng/ml*10 = 1514,55ng/ml. Qua kt qu ta thy nng DNA trong mu thu c l rt cao. Nhng t l 260/280 = 1,0353 < 1,8 chng t lng DNA thu c ny l DNA d, do b nhim mt lng protein. Mu b nhim l do: giai on mu sau khi li tm b tch ra thnh 3 lp, mt lp dch trong ri ti mt lp protein v cui cng l lp c cha chloroform; sau ht phn dch trong pha trn cho qua mt tp mi, nhng trong thao tc ny chng ta u cn chm n mng protein hay lm v mng protein nn c mt lng protein nhim vo mu DNA. Hay do trong qu trnh ta DNA bng isopropanol -20oC protein cng ta theo lm cho mu DNA b nhim protein. BI 2: K THUT PCR * Mc ch: Xc nh tnh trng thm ca la. 1. Nguyn tc ca phn ng PCR: PCR l k thut x l in vitro cc chui m ha di truyn DNA bng cch pht trin primer mt cch ng lot trn cc dy n DNA. Ton b tin trnh c hon thin do s bin cht ca DNA cn thit, s tc ng ca primer ti u ca cc

dy n DNA ny, k n l s pht trin ca cc primer do phn ng DNA polymerase. 1.1.Phn ng PCR gm nhiu chu k, mi chu k c 3 giai on: - Thi k bin cht DNA: tch dy i thnh dy n, nhit c thit k l 950C, vi nhit ny DNA s to ra si n DNA, hot ng nh dy nn cho qu trnh tng hp DNA mi. - Thi k lai gia primer v DNA: Nhit hay i trong khong 50 600C. Nhit ny ty thuc vo loi marker m chng ta ang s dng, vi chiu di c th ca oligonucleotide. Chng ta phi th tng bc bit chc chn nhit cn c thit k l bao nhiu. giai on ny cc on mi s bt cp vi mch n ADN khun mu v tr b sung. y l giai on quan trng nht, bi v trong giai on ny, s kin lai DNA DNA xy ra. Nu nhit qu cao, khng lai c DNA. Nu nhit qu thp, vic lai DNA s b nhiu li. Chnh v th thit lp nhit cho chu k PCR, ngi ta xc nh nhit nng chy Tm vi tng nhm priner nh sau: Tm = 4 x (G + C) + 2 x (A + T) 0C. Trong phn ng PCR ny, chng ta s dng nhit l 580C. - Thi k ko di dy DNA mi nh Tap polymerase: nhit 720C l nhit ti u cho nhng hot ng ca ADN polymerase. Di s xc tc ca ADN polymerase nhng on mi bt cp vi ADN khun mu s c ko di. Cc on ADN mi c hnh thnh li c s dng lm khun tng hp cho cc chu k tip theo. Nh vy s lng bn sao s tng gp 2 sau mi chu k v sau n chu k, tnh theo l thuyt s lng bn sao l 2n 1.2.Phn ng PCR yu cu: - ADN khun mu. - Hai on mi c hiu c chiu di t 15 30 nucleotit.

+ Primer xui: Bt cp vi mch khun ca DNA v s ko di theo chiu phin m. + Primer ngc: Bt cp vi mch m ca DNA v s ko di ngc theo chiu phin m. - Bn loi deoxyribonucleotit triphosphat (dATP, dTTP, dGTP, dCTP). - Enzym ADN polymerase chu nhit. - MgCl2. - Dung dch m. 2. Thnh phn cho phn ng PCR gm: 1.Nc ct. 2.Dung dch m. 3.dNTP 4.Mi 1: EP 5.Mi 2: ESP 6.Mi 3: IFAP 7.Mi 4: INSP 8.MgCl2: 3mM 9.BSA 10.Taq-polymerase: 1U 11.DNA la. 3. Chng trnh chy PCR 950C 2 min 950C 5 sec bt hot protease v cc nuclease bt li. gim thi gian bo ton enzym v DNA khun.

580C 5sec 720C 5sec 720C 5 min 100C forever. 4. Phng php:

35 chu k

Tr mu sau khi hon thnh qui trnh PCR.

Sau khi phn ng kt thc, ly 10ml sn phm PCR in di kim tra trn gel agarose 1,5%. Mi nhm thc hin 6 phn ng + 1 phn ng ph. Thc hin vi 3 mu: + Mt mu i chng dng: gm 2 tp. + Mt mu DNA la: gm 2 tp. +Mt mu i chng m: gm 2 tp. * Trc tin ta trn Master mix cho 7 phn ng. Cc thnh phn ln lt c cho vo mt tp theo th t v hm lng nh sau: 1.Nc ct. 2.Dung dch m. 3.dNTPs 4.Mi 1: EP 5.Mi 2: ESP 6.Mi 3: IFAP 7.Mi 4: INSP 8.MgCl2: 3mM 42ml 17,5ml 28ml 14ml 14ml 14ml 14ml 21ml

9.BSA 10.Taq-polymerase: 1U

1,75ml 1,75ml

Sau khi cho cc thnh phn vo, ta lc u cho hn hp ha ln vo nhau. Sau , ht vo 6 tp nh (6 tp ny c t trong nc ) mi tp 24ml dung dch Master mix. + 2 tp s dng cho i chng dng. + 2 tp s dng cho DNA la. + 2 tp s dng cho i chng m. 2 tp i chng dng ta cho vo mi tp 1ml mu i chng dng, 2 tp DNA cho vo mi tp 1ml DNA la, 2 tp i chng m cho vo mi tp 1ml nc ct. Cc tp ny c trong nc . Sau khi cho y cc mu i chng vo cc tp ta tin hnh chy PCR v chy in di cc mu. 5.Qu trnh in di trn gel agrarose: Phng php in di trong gel agarose l phng php thng dng dng tch ri cc on nucleic c kch thc t 200bp n 50kb. 5.1.Nguyn tc: - Nguyn tc ca phng php ny l da vo tnh phn cc ca phn t v kh nng di chuyn v mt hng xc nh khi chu tc ng ca mt in trng. Trong cc dung dch kim v trung tnh, cc i phn t nucleic acid tch in m ng u trn khp b mt nn trong in trng, chng s di chuyn v cc dng ca in trng - Agarose l loi polyme tch t rong bin, c cu to bi 2 monome l t D-galactose v 3,6-anhydro L-galactose, nn sau khi un si s to thnh mng li cho php cc phn t khc nhau i qua tu theo kch thc v trng lng phn

- Cc on DNA kch thc khc nhau s chy vi tc khc nhau trn gel: on ngn chy xa, on di chy chm. ADN tng s c kch thc ln nht nn chy chm v chim v tr ln nht trn bn gel 5.2.Quy trnh in di: + Chun b gel: Khay in di c t lc sn mt u khay. S dng lc c s rng l 17. Khay c t ni bng phng. Gel s dng chy in di : 40ml vi nng 1,5% agrarose. Trc tin cn 0,6g agrarose cho vo chai thy tinh, sau cho vo chai 50ml Bufer TE, tr hao trong qu trnh nu bc hi ht 10ml. Lc chai cho agrarose ha tan vo Buffer TE, sau em nu trong microwave trong 2 pht 450W (nu cho agrarose ha tan hon ton v trong nh nuc), agrarose sau khi nu xong ngui khong 60oC ri nh dung dch vo mt u khay dung dch s t tri u khay. Khong 45 pht sau gel c li, ta dng tay ly lc ra nhng phi cn thn trnh b b gel. t khay vo b in di, thm dung dch in di ph mt gel khong 2mm. Sau khi gel chun b xong, ta tin hnh load mu vo cc ging ca gel + Load mu: u tin ht dung dch loading buffer (mt dung dch ti DNA cha 30% glycerol, 0,25% bromophenol blue (BPB) v 0,25% xylencyanol (XC), trong thnh phn c cha glycerol c t trng cao s ko DNA nm trong ging khng b tri ra ngoi trong khi load mu) v nh thnh tng git nh khong 2ml ra giy paraflim, sau ht 10ml dung dch mu i chng dng trn iu vi dung dch loading buffer v tin hnh load mu vo ging ca gel. Bt u load t ging th 2, ging th nht dng load thang chun. Tip tc load mu DNA la vo ging k tip v mu i chng m load sau cng.

Sau khi load xong ta y np b in di li v tin hnh chy in di 25V. Thi gian in di thay i ph thuc vo ln ca DNA, nng agarose ca gel v cng dng in. Sau khi chy in di xong bn gel c ly nh ra khi khun gel v ngm vo dung dch EtBr trong thi gian 30 pht c tc dng nh du phn t DNA, ha cht ny c kh nng gn xen vo gia cc base ca nucleic acid v s pht hunh quang di tc dng ca tia t ngoi. Sau ra sch bn gel bng nc ct. Quan st v chp hnh: Bn gel c quan st di nh sng t ngoi rad. Quan st thy ADN hin ln di dng cc vch sng. 5.3.Kt qu: 8 9 10 i chng dng DNA la i chng m mu ADN la l mu la thm ng hp t. Ging Hnh 2. Hnh gel chy in di mu DNA la thm Trn gel in di, ta thy ging 8 c 2 band, c 2 band iu thy r 9 l mu DNA ca la cng th hin 2 band sng, nhn thy r nh mu i chng dng, chng t mu DNA la cng l la thm ng hp t. Mu i chng m ging th 10 l nc ct nn khng th hin band trn hnh chp gel. BI 3: QUY TRNH TRCH DNA T TM I. VT LIU- HA CHT 1. Vt liu sinh hc M tm. 2. Ha cht Tris-HCl (pH8). EDTA. = 260 360 nm trn b c gel bio-

Cloroform. Isoamyalcoohol. 20%SDS. Proteinase K. Ethanol 96% v 70% 7,5M Ammonium acetate. Pha dung dch: dung dch m D gm 100mM NaCl, 10mM Tris.HCl pH 8, 25mM EDTA pH 8, 0.5% SDS, 0.1mg proteinase K. Tr nhit phng. (Thm proteinase K vo ngay trc khi s dng). 3. Dng c Thit b Micropipet. My li tm. Tp Eppendorf 1,5 ml, 2,0 ml T m. a thy tinh. II. PHNG PHP TIN HNH 1. Chun b mu: Tm em bc b v phn lng tm, dng ko ct mt ming tht tm cho vo tp 2,2ml. Sau , dng a thy tinh nghin mu thnh bt mn. 2. Trch DNA: Bc 1: ph v mng t bo: Mu sau khi nghin mn cho thm vo tp 400ml dung dch trch buffer D gip ph v mng t bo, lc u v qua m nhit 50oC trn my lc vi vn tc 500rpm.

Bc 2: loi b protein v cc thnh phn hu c: Sau khi xong, ta cho thm vo tp 400ml dung dch Chloroform:isoamylalcohol (24:1) chit xut protein v cc thnh phn hu c khc (polysaccharides, polyphenols, lipids,), lc nh ri em ly tm vi vn tc 13000 vng/pht trong thi gian 10 pht. Khi li tm xong tp phn ra thnh 3 lp, lp th nht l phn dch trong trn cng c cha DNA, lp th hai l mt lp protein, lp k tip c cha chloroform v cc thnh phn hu c khc. Bc 3: ta DNA: Ta tin hnh ht 300ml phn dch trong pha trn cho qua mt tp 2,2ml mi, sau thm vo tp ny 200ml 7,5M ammonium acetate v 800ml ethanol 96% c tc dng gip DNA kt ta, bo v chng khi s phn hy ca cc enzyme v ra sch DNA. Lc nh v tin hnh li tm vi vn tc 13000 vng/pht trong 5 pht. Bc 4: ra DNA: Sau khi li tm xong, ta loi b phn dch trong trn ly phn cn pha di. Ra phn cn vi 800ml ethanol 70% 2 ln loi b mui ra khi DNA. Ly tm 13000 vng/pht trong 5 pht sau mi ln ra. b ethanol sau mi ln ra. Bc 5: sy kh DNA: Sy kh bng my ly tm chn khng nhit 45oC trong 15 pht, trnh trng hp mu DNA cn cha ethanol s nh hng n DNA tng s. Bc 6: ha tan DNA: Sau khi sy mu xong, ht 30ml nc ct cho vo tp cha DNA ha tan DNA. Sau ht 10ml dung dch ny cho vo tp 1,5ml mi ht sn 90ml nc ct, lc u. Sau em mu o OD. 3. Phng php o OD: 3.1.Nguyn tc: - Nguyn tc ca phng php ny l da vo s hp th mnh nh sng ca mt cht mt bc sng xc nh. Nucleic acid hp th mnh nh sng t ngoi bc sng 260 nm do s c mt ca base purine v pyrimidine. Gi tr mt quang bc sng 260nm (OD260nm) ca cc mu cho php xc nh nng nucleic acid trong mu. . n v OD260nm tng ng nng 50ng/ml cho mt dung dch ADN si i.

Do nng ADN trong mu c tnh theo cng thc sau: CADN (ng/ml) = OD260nm * 50 * pha long - kim tra sch ca dung dch, ngi ta o thm gi tr OD 280 nm (OD280nm). bc sng ny, cc protein c mc hp th cao nht. Ngoi ra, cc protein cng hp th nh sng bc sng 260 nm nh cc nucleic acid v do lm sai lch gi tr tht ca nng nucleic acid. Mt dung dch nucleic acid c xem l sch (khng tp nhim protein) khi t s : OD260nm/OD280nm nm trong khong 1.8 2 3.2.Cc bc o OD: - Ly 100ml nc kh ion v trng vo cuvet 100ml, o i chng. - Ho 10ml dch ADN cn o vo 90ml nc kh ion v trng, bm vo cuvet, o bc sng 260nm, 280nm - c hp th trn my o quang ph bc sng 260 nm, 280 nm. X l s liu theo cng thc trn. 3.3.Kt qu o OD: Sau y l bng th hin kt qu o OD ca mu Tm: 260nm 1,8202 280nm 0,9305 260/280 1,9561 280/260 0,5112

Ta c: OD260nm = 1 DNA = 50ng/ml Qua kt qu o OD ca Tm ta c: OD260nm = 1,8202 Trc khi tin hnh o OD, mu c pha long 10 ln. Vy nng DNA trong mu Tm thu c: CDNA= OD260nm * 50ng/ml*10 = 1,8202*50ng/ml*10 = 910,1ng/ml.

Qua kt qu ta thy nng DNA trong mu thu c l tng i. Nhng t l 260/280 = 1,9561 nm trong khong 1,8 2 chng t lng DNA thu c ny l DNA sch. BI 4: TRCH DNA T VI KHUN GRAM M I. VT LIU- HA CHT 1. Vt liu sinh hc Vi khun Escherichia coli 2. Ha cht Tris-HCl (pH8). EDTA. Isopropanol. 10% SDS. Chloroform. Ethanol 96% v 70%. Proteinase K. 3. Dng c Thit b Micropipet. My li tm. Eppendorf 1,5ml v 2ml. II. NGUYN TC Vic thu nhn DNA t t bo vi khun c tin hnh thng qua nhiu bc. Cc giai on c bn ca tch chit DNA bao gm: (1) Ph mng t bo v mng nhn, (2) Loi protein, (3) Ta DNA.

Tch chit DNA T bo vi khun c ha tan vo dung dch SDS v proteinase K. + Proteinase K l mt serine protease khng c bit, c s dng chit xut v tinh sch DNA c trng lng phn t cao, ngoi ra proteinase K cng c dng chit xut ARNase v lm bt hot nuclase (ARNase v ADNase). hot ng c hiu qu hn Proteinase K thng c duy tr trong s hin din ca SDS, EDTA v ure. tch protein ra khi DNA, phenol hoc chloroform c s dng cho mc ch ny. III. PHNG PHP TIN HNH 1.Chun b mu: Vi khun Ecoli c nui qua m. 2. Trch DNA: Bc 1: loi b cc thnh phn tp nhim: Cho vo 2 tp (2,2ml) mi tp 2ml dung dch vi khun Ecoli c nui cy qua m. Sau , li tm 13000rpm trong 5 pht. Li tm xong loi b phn trong ly phn ta pha di. Bc 2: ha tan DNA: Bm 250ml TE vo tp th nht ha tan kt ta tp th nht, sau chuyn phn dung dch tp th nht sang tp th hai, ha tan DNA tp th hai. Bc 3: chit xut DNA: Sau , cho thm vo tp 50ml dung dch 10% SDS cng 5ml Proteinase K lc u, 20 pht nhit 65oC, mi 5 pht o ngc tp mt ln trn u dung dch, c tc dng ph v mng t bo v mng nhn, chit xut v tinh sch DNA. Bc 4: to phc vi CTAB: Sau khi ha tan ht ta ta cho thm vo tp 400ml CTAB v 65oC trong 20 pht, khong 5 pht o ngc ng tp trn u. Phng php ny s dng CTAB (Hexadecyltrimethylammoniumbromid) mt cht ty cation, ha tan mng t bo lm cho DNA d ha tan hn, bin tnh protein v hnh thnh phc hp vi nhng

polysaccharide, protein, polyphenol,. CTAB c kh nng lin kt thun nghch vi ADN v polysaccharide, c th nng mui cao (trn 1.4 M NaCl c trong m chit) th CTAB kt hp vi ADN v polysaccharide nhng phc hp CTAB-ADN tn ti trng thi ha tan, trong khi phc CTAB-polysaccharide li kt ta -phc hp ny sau c tch ra khi dung dch tch chit bng hn hp chloroform isoamyl alcohol. Bc 5: loi b protein v cc phc hp: Tip tc thm 600ml chloroform:isoamylalcohol (24/1), lc u v li tm 13000 vng trong 10 pht loi b protein, nhng polysaccharide v cc phc hp trn. Bc 6: ta DNA: Li tm xong, chuyn 500ml phn trong pha trn vo tp mi v thm 1ml isopropanol lc u, gi 20oC trong 30 pht nhm mc ch ta DNA, khng cho DNA bin tnh, thu c DNA nhiu hn v bo v chng khi s phn hy ca cc enzyme. Tin hnh li tm 13000 vng trong 10 pht tp trung lng DNA kt ta xung y tp. Bc 7: loi b phn dch trong: Sau khi li tm, ta loi b phn dch trong pha trn v thu phn kt ta pha di. Bc 8: ra ta: Ra ta vi 1ml ethanol 70% ly tm 13000rpm trong 5 pht, lm 2 ln loi b mui bm trong DNA v loi b lng isopropanol cn li trong DNA (mi ln b ethanol). Bc 9: phi kh DNA: Ra DNA xong, ta tin hnh phi DNA qua m nhit phng cho sch ht ethanol trnh nh hng n DNA tng s. Sau , cho vo tp 100ml nc ct, lc u cho DNA tan ra v tin hnh o OD. 3.Phng php o OD: 3.1.Nguyn tc: - Nguyn tc ca phng php ny l da vo s hp th mnh nh sng ca mt cht mt bc sng xc nh. Nucleic acid hp th mnh nh sng t ngoi bc sng 260 nm do s c mt ca base purine v pyrimidine. Gi tr mt quang bc sng 260nm (OD260nm) ca cc mu cho php xc nh nng nucleic acid trong mu. . n v OD260nm tng ng nng 50ng/ml cho mt dung dch ADN si i.

Do nng ADN trong mu c tnh theo cng thc sau: CADN (ng/ml) = OD260nm * 50 * pha long - kim tra sch ca dung dch, ngi ta o thm gi tr OD 280 nm (OD280nm). bc sng ny, cc protein c mc hp th cao nht. Ngoi ra, cc protein cng hp th nh sng bc sng 260 nm nh cc nucleic acid v do lm sai lch gi tr tht ca nng nucleic acid. Mt dung dch nucleic acid c xem l sch (khng tp nhim protein) khi t s : OD260nm/OD280nm nm trong khong 1.8 2 3.2.Cc bc o OD: - Ly 100ml nc kh ion v trng vo cuvet 100ml, o i chng. - Ho ADN cn o vo 100ml nc kh ion v trng, bm vo cuvet, o bc sng 260nm, 280nm - c hp th trn my o quang ph bc sng 260 nm, 280 nm. X l s liu theo cng thc trn 3.3.Kt qu o OD: Sau y l bng th hin kt qu o OD ca mu vi khun Ecoli: 260nm 0,1140 280nm 0,0555 260/280 2,0524 280/260 0,4872

Ta c: OD260nm = 1 DNA = 50ng/ml Qua kt qu o OD Tm ta c: OD260nm = 0,1140 Trc khi tin hnh o OD, mu vi khun khng tin hnh pha long 10 ln. Vy nng DNA trong mu vi khun Ecoli thu c: CDNA= OD260nm * 50ng/ml = 0,1140*50ng/ml= 5,7ng/ml.

Qua kt qu ta thy nng DNA trong mu thu c mc d l rt thp nhng qua t l 260/280 = 2,0524 nm trong khong 1,8 2 chng t lng DNA thu c ny l DNA sch. Cu hi 1. Chng ta c th s dng knh hin vi thy DNA trch khng? Ta ch thy l mt khi ng nht acid nucleotid v kch thc chui polypeptide rt nh nn khng th nhn bng knh hin vi thng thng. 2. Phng php trch DNA hu dng nh th no i vi ngi nghin cu? Trch ADN gip xc nh c trnh t cc nucleotid trn cc gen, t xc nh c cc tnh trng ng dng trong chn ging cy trng, sn xut vaccin phng v tr mt s bnh di truyn ng vt, ngi Kiu gen c tnh di truyn phn nh mi quan h huyt thng trong chng tc, gia nh, xc nh c chnh xc c th rt hu dng trong cng tc nhn din nhn thn. 3. Theo bn s lng nhim sc th c nh hng n khi lng DNA thu hoch c sau khi li trch khng? C nh hng, v ADN nm trn nhim sc th nn s lng nhim sc th t l thun vi khi lng ADN. 4. Qui trnh trch DNA ca l bi bn ang s dng c th dng trch DNA ca l la khng? Nhn chung b gen ca cc loi thc vt khng khc nhau nhiu v cu trc, nn chng ta c th dng quy trnh tch chit ADN l cy h cam,qut cho cy la mc d quy trnh khng c ti u hon ton cho loi cy . BI TP Bi tp1: Pha 100ml dung dch EB (0.1M Tris.HCl pH 8; 0.5M NaCl, 0.05M EDTA pH 8; 0.01% -mercaptho ethanol) t cc dung dch gc Tris.HCl 1M, NaCl 5M, EDTA 0.5M; -mercaptho ethanol 100%. Phi mt bao nhiu dung dch gc pha dung dch EB. Da vo cng thc C1V1=C2V2 ta tnh c cc th tch cc dung dch gc cn pha nh sau: V1 = C2V2 / C1

* VTris.HCl = 0.1*100/1 =10 ml * VNaCl = 0.5*100/5 = 10ml * VEDTA = 0.05*100/0.5 = 10ml * V -mercaptho ethanol = 0.01%*100/100% = 0.01ml Tng th tch ca dung dch gc = 10 + 10 + 10 +0.01 = 30.01ml Th tch nc ct thm vo = 100 30.01 = 69.99 ml Bi tp 2: in vo tt c cc trng trong bn sau y. Cho bit mch no l mch m, mch no l mch khun. Ghi u 5 v 3 ca DNA v u NH2, COOH ca protein. Phn t Mch :khu n A D N Mch :m 5 5 5 G G U C C G A A C A A G A A U C C U T U U G G C A A A C C A G G C T U G 3 3 3 3 C G T T T G A C T G C A 5 u Trnh t u

mARN Anticodon

ca tARN COO Acid amin H2 N Ala N Stop Arg H

Anticodon v codon bt cp b sung theo hnh thc i song song Th d: 5 AUG 3 th anticodon phi l 3 UAC 5

Bi tp 3: in vo tt c cc trng trong bn sau y. Cho bit mch no l mch m, mch no l mch khun. Ghi u 5 v 3 ca DNA v u NH2, COOH ca protein. Phn t Mch:kh un AD N mARN Mch:m 5 5 A A T U G G A A A A C C T U G G A A C C G G T U 3 3 3 T A C T T G A C T G C A 5 u Trnh t u

Anticodon ca tARN 5 H2 Acid amin N Met N Stop Arg C A U G U U U C A A C G 3 CO OH