TRNG I HC M THNH PH H CH MINH KHOA CNG NGH SINH HC

NHP MN CNG NGH SINH HC

Bi bo co:

IN DI GEL AGAROSE V NG DNG

Ging vin

TS V Minh Tr Phan Duy Lun MSSV: 0853010470 on Th Thin Trang MSSV: 0853010961

Danh sch nhm:

TP H Ch Minh, ngy 28, thng 10, nm 2010

MC LC
I. GII THIU CHUNG 1.in di 2. Phn loi 3. Nguyn tc thc hin II. IN DI TRN AGAROSE GEL 1.Gii thiu v agarose 2. Agarose gel 3. Cac yu t anh hng n tc dich chuyn in di trong agarose gel 3.1. Kich thc cua phn t 3.2. Nng agarose 3.3. Cu hinh cua DNA 4.Thnh phn thc hin in di 4.1. Agarose 4.2. Lc 4.3. Khun gel 4.4. My in di 5. Phng phap in di agarose gel 5.1. m pH 5.2. Chun bi agarose gel 5.3. Nhum DNA trong agarose gel 5.4. Thu hi DNA t agarose gel 5.4.1. Dng agarose c nhit nng chy thp (low melting temperature) 5.4.2. Ly tm 5.4.4. Dng ht thy tinh 5.4.3. in di vo by 6. Cc bc thc hin 6.1. Chun b gel agarose 6.2. Tin hnh in di 6.3. Nhum DNA bng EtBr 6.4. Kim tra bng AND III.NG DNG
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1. Nhng trng hp s dng in di gel agarose 2.Tinh sch v thu nhn mu 3. nh tnh v nh lng th nucleic acid 4. Mt s v d I. GII THIU CHUNG 1.in di: - L hin tng dch chuyn cc vt th mang in tch di tc ng ca in trng. S dch chuyn ny do phn lc in trong lc Lorentz. in di trn gel (gel electrophoresis) p dng trong sinh hc phn t DNA, RNA, Protein da trn cc c im vt l ca chng nh kch thc, hnh dng hay im ng tch (isoelectic point). K thut ny s dng mt dung dch m (buffer) dn in v to in trng u. Mt bn gel ng vai tr l th nn phn tch cc phn t, v cc cht cht nhum khc nhau (ethumdromide, xanh coomassie, bc) pht hin v tr cc phn t gel sau khi in di. - in di la ky thut c dng phn tch v i khi tinh sch cac i phn t c bit l cc protein v cac nucleic acid trn c s kch thc khi lng, in tich v cu hnh cua chung. - Khi cc phn t tch in c t trong mt in trng, chng s dch chuyn hng n cc dng (+) hoc cc m (-) ty theo in tch ca chng. Ngc vi protein, loi phn t c in tch thc hoc dng hoc m, cc nucleic acid c mt in tch m khng i nh khung phosphate ca mnh, v v th ch dch chuyn hng n cc dng. - Cc phn t protein v nucleic acid c th c chy in di trn mt khun (support matrix) nh giy, cellulose acetate, gel tinh bt, agarose hoc polyacrylamide gel. Trong gel ca agarose v polyacrylamide c s dng ph bin nht. Thng thng, gel l mt khun c dng phin mng c cc ging np (loading) mu. Gel c ngm trong m in di cung cp cc ion dn truyn dng in v mt vi loi m duy tr pH mt gi tr khng i tng i. 2. Phn loi Gel c cu to bi cc chui cao phn t c lin kt cho vi nhau to thnh mt h thng mng li vi kch thc cc mc li ty thuc vo nng cht cao phn t v phn ng to lin kt cho. Cc kiu in di: in di trn gel agarose: kh nng phn tch gel 50bp 20kb in di trn gel polyacrylamide : kh nng phn tch gel 5bp 1kb Ngoi ra cn in di trong trng xung (PFGE _ Pulse Field Gel Electrophonesic)

3. Nguyn tc thc hin - K thut in di hot ng nh vo lc ko ca in trng tc ng vo cc phn t tch in v kch tht l ca th nn (gel). - Cc phn t m hay dng trong mt in trng s di chuyn trong gel vi vn tc khc nhau nh vo s khc nhau ca: Lc in trng tc ng ln chng(nu cc phn t tch in khc nhau) Kch thc ca phn t so vi kch thc ca l gel Hnh dng, cng knh ca phn t II. IN DI TRN GEL AGAROSE 1.Gii thiu v agarose - Agarose (polysaccharide) c khi lng phn t xp x 120.000 Da l mt trong hai thnh phn chnh ca agarose chim khong 70%, phn kia l agaropectin chim khong 30%. Agarose l mt polymer mch thng khng b sulphate ha cha hai gc xen k nhau l D-galactose v 3,6-anhydro-L-galactose. Agarose gel l mt cht trong sut (transparent) hoc trong m (transluent) ging nh agar, c to thnh khi hn hp agarose v nc (hoc m in di) c un nng ti >100oC v sau c lm lnh; dng gel xut hin khong 40-45oC. Agarose gel c ng dng rng ri lm gi th cho cc nucleic acid trong k thut in di ngang (horizontal electrophoresis) hoc lm gi th cho mi trng nui cy bacteriophage (top agarose). - Agarose c tch ra dng thch t mt s loi bin to, hoc rong bin , c tm thy ti California v min ng chu . Agar, mt thut ng c ngun gc t ch thch agar-Malay, c ngha l sa ong cha, l thng bt ngun t cc chi Gelidium rong bin. N bao gm c phn t agarose v agaropectin v cung cp h tr cho cc thnh t bo trong to bin.Agar c s dng nh mt cht lm c thc n, ging nh gelatin, hoc mt phng tin cho vi khun pht trin, nm, hoc cc vi sinh vt khc.

Hnh : Cu trc phn t ca agarose. n v agarobiose (v d: hai phn t ng) l mt monomer trong agarose polymer. C khong 400 monomer trn mt chui polymer.

2. Agarose gel - Agarose gel l mt loi gel thng dng nht, mt phn do thao tc n gin, thng dng phn tch nhng on c kch thc trong khong 0.5 20 kb. Gel c trn mt gi th nm ngang v c in di c thc hin theo phng nm ngang. - Cac phn t nucleic acid co khi lng va in tich khac nhau c tach ra khi di chuyn t cc m sang cc dng cua h in di trong mt in trng co in th va cng thich hp. Ky thut nay n gian va thc hin nhanh. Hn na, vi tri cua DNA trong gel c xac inh trc tip: cac bng DNA trong gel c nhum nng thp cua thuc nhum huynh quang ethidium bromide (EtBr) v co th pht hin di anh sang t ngoai (ultraviolet-UV). - Mt khc, c lng kch thc cc trnh t nucleic acid trong gel agarose, ngi ta s dng mt yu t nh du trng lng phn t (monocular weigth marker MWN). l mt trnh t DNA c kch thc bit (thang DAN DNA ladder), thng thng ngi ta s dng thang DNA l thc th c thy gii bi enzyme gii hn HindIII 3. Cac yu t anh hng n tc dich chuyn in di trong agarose gel 3.1. Kich thc cua phn t Cac phn t DNA mach thng si i i qua ban gel cac tc ty l nghich vi ham log10 cua khi lng phn t cua chung. Do , cc phn t DNA c kch thc cng ln (khi lng phn t ln) th tc dch chuyn cng chm.

3.2. Nng agarose oan DNA mang kich thc nht inh s dich chuyn cac tc khac nhau qua cac ban gel cha cac nng agarose khac nhau. Nh vy, dung gel cac nng khac nhau co th phn tch c cac oan DNA co kich thc khac nhau). Nng agarose cao c kh nng phn tch cc on DNA nh, trong khi nng agarose thp li cho php phn tch cc on DNA ln hn.

Bang: Cac thng s in di DNA bng agarose gel 3.3. Cu hinh cua DNA Cac DNA dang vong ong (form-I), vong t (form-II) va mach thng (form-III) co cung mt khi lng phn t se dich chuyn trn agarose gel cac tc khac nhau. Nhn chung, DNA ca cc plasmid mch vng dch chuyn nhanh hn DNA ca plasmid cng loi nhng c dng mch thng. Hu ht plasmid mch vng cha t nht hai dng DNA c cu trc khng gian khc nhau l dng vng ng (siu xon) v vng t. 4.Thnh phn thc hin in di 4.1. Agarose Agarose l dn xut polysaccharide t agar (thch) c tinh khit cao. C rt nhiu loi agarose khc nhau v in mi, nhit nng chy, trong, cng. agarose thng c cung cp di dng bt kh. Khi cho vo m si n s tan v gi c trng thi lng cho n khi to gel khi nhit h xung khong 40C. Khi ng gel n nh nhit thp hn. Kch thc mng li v c tnh sn lc ph thuc vo nng agarose trong gel. Nng cng cao, mng li cng nh. Nng thch hp l 0,4% n 4%. 4.2. Lc Dng to ra cc ging trong bn gel 4.3. Khun gel:
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Ni gel ln

Hnh: Thnh phn thc hin in di 4.4. My in di - Bung m c to nn bng cch tch hai bn knh gia hai thanh spacer (to b dy) hai u, sau trm cc u v y gel to bung kn (H. 1.4). Gel bn c kch thc t 2,5 cm2 (gia hai lamen ca knh hin vi) n 30.15 cm2 v dy t <0,05 mm n > 10 mm. - in cc. - My in di

Hnh nh v thit b in di agarose 5. Phng phap in di agarose gel 5.1. m pH Mt s m in di thich hp cho DNA si i thng c dng la Tris-acetateEDTA (TAE), Tris-borate-EDTA (TBE) v Tris-phosphate-EDTA (TPE) nng khoang 50 mM va pH 7,5-7,8. Thng do thoi quen, TAE c s dung nhiu nht, nhng kha nng m cua no lai thp. m TBE v TPE u co kha nng hoa tan tt cac oan DNA va co kha nng m cao hn mt cach ro rt. Cc on DNA s dch chuyn vi cc tc hi khc nhau mt cht trong ba loi m trn do s khc nhau v cng lc ion ca m. m khng nhng thit lp mt gi tr pH, m cn cung cp cc ion h tr cho dn (conductivity). Nu chng ta dng nc thay v l m trong qu trnh in di, th s khng c s dch chuyn cn thit ca DNA trong gel. Ngc li, nu s dng m nng m c (v d: dung dch stock 10), th nhit trong gel c th tng cao v lm nng chy n. 5.2. Chun bi agarose gel Co nhiu loai agarose khac nhau thich hp chay in di, loai agarose dung tt nht trong thi nghim la type-II-agarose co ni thm thu thp (low-endo-osmotic). No d dang chay ra va tao thanh mt dung dich trong sut, kt qua la gel an hi thm chi cac nng thp. Tuy nhin, type-II-agarose d bi bn bi sulphate

polysaccharides (SP), hn na SP con c ch cac enzyme nh ligase, polymerase va RE. Vi th, cac oan DNA dung ly t nhng gel nh th phai c lam sach cn thn trc khi chung c dung nh la cac khun mu hoc c cht cho cac enzyme nay. 5.3. Nhum DNA trong agarose gel Phng phap quan sat DNA trong agarose gel thun li nht la dung thuc nhum huynh quang EtBr. EtBr c dung pht hin DNA si i va RNA si n. Tuy nhin, ai lc (affinity) cua thuc nhum i vi nucleic acid si n la tng i thp va co hiu sut huynh quang khng cao. Sau khi nhum, EtBr s xen vo gia cc base ca nucleic acid v cho php pht hin d dng chng trong gel. Mc du tinh linh ng in di cua DNA si i mach thng giam khi co mt thuc nhum (khoang 15%), nhng kha nng xac inh gel trc tip di anh sang UV trong sut qua trinh in di hoc giai oan cui co u th ro rt. Tuy nhin, nu cn co th chy in di gel khng co EtBr va chi nhum DNA hoc RNA sau khi in di xong. 45Gel c ngm trong m in di hoc H2O cha EtBr (0,5 phut nhit phong). Chu y : Ethidium bromide la mt tac nhn gy t bin manh, cht c gy ung th, v vy cn lun lun eo gng tay khi cm gel hoc dung dich cha thuc nhum,trnh tip xc trc tip vo c th v phi c ni ng cht thi ring sau khi s dng, khng c thi ra ngoi mi trng khi cha c x l thch hp.

Cu trc ethidium bromide 5.4. Thu hi DNA t agarose gel Nhiu phng phap a c xy dng thu hi DNA t agarose gel, nhng khng co phng phap nao la hoan toan ti u. Co hai vn chinh: th nht a s cac loai agarose u bi nhim bn bi sulphate polysaccharides, cac san phm sau o c chit t gel cung vi DNA, chung la nhn t c ch co hot tnh cua nhiu loai

enzyme (RE, ligase, kinase, polymerase) dung trong cac bc tao dong tip theo sau; th hai hiu sut chit DNA t agarose gel phu thuc vao khi lng phn t cua no, cac oan DNA co chiu dai <1 kb co th c thu hi hon ton, khi khi lng phn t cua DNA tng thi hiu sut chit giam, cac oan DNA co kich thc >20 kb c thu hi kem (khoang hn 20% san lng). C nhiu phng php thu hi v tinh sch DNA t agarose gel, di y l mt vi phng php chnh: 5.4.1. Dng agarose c nhit nng chy thp (low melting temperature) Mu agarose gel c nhit nng chy thp cha on DNA quan tm c ct ra khi bn gel v cho vo trong m vi t l 1:1, b sung mui n nng 0,5 M, sau c nng chy 70oC ha tan hon ton trong m. Dung dch gel c DNA c chit bng phenol/chloroform v kt ta. Lu DNA c tch chit bng phng php ny khng thch hp cho mi phng thc thao tc tip theo, do s hin din ca cc nhn t c ch trong agarose. 5.4.2. Ly tm Mu agarose gel c cha bng DNA quan tm c ct ra v cho vo m chit, lm nng ha tan hon ton, sau cho dung dch gel ln trn mt lp (mng) bng thy tinh silicon ha t trong mt ct lc nh loi 0,5 mL (cn gi l spin column). Ct ny c cho vo mt tube vi ly tm loi 1,5 mL v c ly tm tc cao 10.000-15.000 rpm trong 10-15 pht. DNA s lin kt vi mng cn dung dch m s i qua lp bng thy tinh vo tube vi ly tm. Cho m ra vo ct v ra ct vi ln bng cch ly tm nhanh. Thay tube vi ly tm mi, cho m ha tan DNA vo ct v ly tm dung ly (elution) DNA ra khi mng vo tube. Dung dch DNA thu c c th c tinh sch thm nu cn. Cng c th ng lnh mu gel trc khi dng phng php ly tm ci thin hiu sut thu hi DNA. 5.4.3. in di vo by Mt ci rnh nh c ct trong agarose gel ngay pha trc ca bng DNA quan tm. Rnh ny c lm y bng glycerol v gel c in di nhanh tr li chuyn DNA t gel vo trong dung dch glycerol (qu trnh in di c th c kim sot bng UV). Dung dch glycerol-DNA sau c chit bng pipette. Hoc sau khi in di, mt khe nh c ct trong gel ngay pha trc bng DNA quan tm v t trong khe mt mu giy loi NA-45. Gel sau c in di tr li v bng DNA quan tm s dch chuyn vo trong mu giy. Mu giy sau c ra v DNA c dung ly trong m bng cch un nng mu giy ti 70oC. DNA sau c th c tch chit bng phenol v kt ta. 5.4.4. Dng ht thy tinh

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Mu gel quan tm c ha tan trong dung dch mui NaI (mt loi chaotropic salt) nng khong 4M. Cc ht thy tinh sau c b sung vo dung dch lin kt hiu qu vi cc on DNA c phng thch nng cho ca NaI. RNA, protein v cc tp cht khc khng lin kt vi cc ht thy tinh. Tip theo l cc chu k ra v kt ta tiu th, DNA tinh sch c dung ly khi ht thy tinh trong m mui thp. Tuy nhin, phng php ny mc d nhanh v hiu qu nhng li c phm vi ti u hp. Thu hi cc on DNA nh (500-800 bp) l khng hiu qu lm v cc on ln (>15 kb) c th lin kt vi cc ht thy tinh khc nhau v cc si DNA c th b ph v trong sut cc chu k ra. 6. Cc bc thc hin

6.1. Chun b gel agarose - Cho lng thch hp agarose v dung dch m vo bnh tam gic. - Gia nhit bng cch hp 5 pht trong ni hp cao p 115 C hoc bng vi sng. Ch khi dng l vi sng cn quan st trnh tro gel ra ngoi, nn ch n khi gel

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si th ct in, ly ra (coi chng bng) lc u ri cho vo lm si ln na. Lp li ba ln cho agarose tan hon ton. - Cho vo chu bo n ~50C cho n khi dch gel t n khong 50 60 C (tay chm vo c) th cho vo gel mt lng dung dch ethidium bromide c hm lng cui cng ca cht mu ny l 50 g/ml. (Nu khng cho ethidium bromide vo gel lng trc khi rt gel vo khun th ngm bn gel vo dung dch ny sau khi in di). - Rt gel vo khun c chn hai u bng bng dn hoc bng kp, ci sn lc v t trn mt phng - Ch cho n khi gel rn hon ton th cn thn tho lc ra khi gel. 6.2. Tin hnh in di - Tho bn gel ra khi kp hoc bng chn, t khun gel trong chu in di ngang, rt dung dch m vo cho ngp gel, ch u c l lc nm pha cathode. - Pha nucleic acid cn in di vi dung dch mu ti mu (in di) 6 hoc bng hp cht mu tng t. Dch mu c t trng cao ny gip nucleic acid khng b xo ng bi dng i lu ca dung dch nn khi ti vo l rng lc th nm gn trong v c mu r rng. Dung dch mu ti mu 6 (dch np mu) cha 30% glycerol, 0,25% bromophenol blue (BPB) v 0,25% xylencyanol (XC). - Bng micropipet ht mu DNA nh vo l rng lc.

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- Bt in mt chiu thc hin in di. Cng dng in khong 50 n 150 V ty loi thit b in di. Thi gian in di thay i ph thuc vo ln ca DNA, nng agarose ca gel v cng dng in. Ch lng DNA c th in di trong mi l rng lc nhiu t ty kch thc rng lc, nu qu nhiu s xut hin hin tng "tailing" (ko ui) kh phn li.

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6.3. Nhum DNA bng EtBr Sau khi chay in di xong ly nhe ban gel ra khoi khun va ngm vao dung dich EtBr nng 0,5 gian 30-45 phut, tt nht trn mt may lc nhe ( 10 vong/phut ). dich nhum EtBr vao mt binh ring x ly va ra ban gel bng cach ngm trong nc ct hai ln, mi ln 15-20 phut. EtBr thng pha sn dang m c 5 mg/mL va gi 4oC trong ti. 6.4. Kim tra bng ADN - Ban gel sau khi nhum EtBr c quan sat di anh = 302 nm. Dung thit bi chuyn dung phn tch v lu tr hnh nh DNA (Gel Documentation System). Chu y: khng nhin vao anh sang t ngoai nu khng co kinh loc thich hp. - Nu in di DNA trong gel c pha sn ethidium bromide th chiu tia t ngoi (UV) nucleid acid s hin hnh di dng nhng vch mu cam.

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- Nu khng cho trc ethidium bromide th sau khi in di cn ngm gel 30 - 60 pht trong dung dch thuc nhum ny nng 0,5 g/ml. - Nu cn chp nh lu t liu th c th dng my nh pollaroid vi ng knh c phin lc nh sng thch hp vi film pollaroid 667, hoc dng thit b phn tch gel s ha (gel documentation system). Ch : n t ngoi (UV) c hai loi bc x vi bc sng 254 nm (UV sng ngn) v 366 nm (UV sng di). UV sng ngn gip quan st r DNA nhng lm tn hi cu trc cht ny, cho nn nu cn thu hi DNA t gel th khng nn p dng

H thng chp nh in di geldoc XP ca Biorad

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A. Kt qu in di trn gel agarose chp bng nh sng UV B. Kt qu Western blot chp bng nh sng trng III. NG DNG - Agarose gel in l mt phng php s dng rng ri vic chia tch cc phn t trn c s tnh ph in, kch thc v hnh dng. Phng php ny c bit hu ch trong vic tch cc phn t sinh hc tnh quan trng nh DNA (deoxyribonucleic acid), RNA (ribonucleic acid), v protein. - Agarose gel l mt cht c s dng trong khoa hc cho in gel v sc k loi tr kch c. S dng agarose gel tch v phn tch protein v DNA . Phng tin bao gm mt bt agarose tinh khit c un si trong mt dung dch m v sau lm lnh. 1. Nhng trng hp s dng in di gel agarose: - c lng kch thc ca cc phn t DNA sau khi thc hin phn ng ct hn ch (v d: lp bn hn ch ca DNA c to dng...). - Phn tch cc sn phm PCR (v d: trong chn on di truyn phn t hoc in du di truyn...). - Phn tch DNA h gen c ct hn ch trc khi thm tch Southern, hoc RNA trc khi thm tch Northern.

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u im ca phng php ny l gel c rt d dng, khng gy bin tnh mu, v bn vng vt l hn polyacrylamide. Mu cng d thu hi. Nhc im l agarose gel c th b nng chy trong qu trnh in di, m c th b tiu hao, v cc dng khc nhau ca nucleic acid c th chy khng n nh. 2. Tinh sch v thu nhn mu Nguyn tc: Da vo s thay i hng in trng trong qu trnh in di. Mi ln hng in trng thay i th phn t DNA phi t nh hng li. Sau khi in di cc vch tng ng vi DNA cn tinh sch c pht hin v thu nhn li theo mt trong nhng phng php: Phng php 1: phn agarose cha cc vch c ct ra v DNA c thu nhn sau khi khuch tn phn t gel agarose vo mt dung dch m thch hp. Phng php 2: mt ging nh c khot trong agarose ngay trc vch DNA. ging ny c bm y dung dch m v in trng c ti lp. DNA di chuyn vo ging cha y dung dich m v c thu nhn li. Phng php 3: in di c thc hin trong mt gel agarose c bit (nh Nusieve hay Seaplaque) c im nng chy rt thp (65). Sau in di vch DNA c ct ra, trong mt dung dch m 65. Khi agarose hon ton tan chy, DNA c thu nhn li sau nhiu cng on tch chit v ta. 3. nh tnh v nh lng th nucleid acid Sau khi thu nhn nucleid acid dng sch, ngi ta tin hnh phn tch nh tnh v nh lng chng: nh tnh: s hin din, cu hnh phn t, kch tht nh lng: hm lng tng i so vi thang hm lng 4. Mt s v d 4.1. V d 1: in di trn gel agarose 1% kim tra ADN tng s tch t cc chng B. THURINGIENSIS var. kurstaki phn lp: t knh (1-10) theo th t (47S, 50S, H22, H15, 29S, T58, H8, H3, 28S, H13 ).

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Qua nh in di cc mu ADN tng s tch c t 10 chng B. thuringiensis kurstaki phn lp u c mt di bng sng tp trung r nt pha trn ca bn gel, khng c cc vch ph, cc mu ADN tch c l nguyn vn v khng b t gy, m bo cht lng cho cc nghin cu tip theo. 4.2.V d 2: in di trn gel agarose 1% kim tra cc plasmid ti t hp mang sn phm PCR c hiu ca gen cry1Ab ct bng EcoR I. Ch thch: Knh 1: Sn phm PCR ca on ADN c hiu ca gen cry1Ab Knh 2,3,5: Plasmid ct bng EcoR I Knh 4: Slasmid ca khun lc xanh Knh 6: Ch th phn t (ADN ct bng Hind III v EcoR I , t trn xung 21226, 5148, 4973, 4277, 3530, 2027, 1904, 1584, 1330, 983, 831, 564 bp). Kt qu hnh cho thy khi plasmid ti t hp c ct bi enzym EcoR I on ADN c hiu ca gen cry1Ab c tch ra khi plasmid c kch thc ng bng vi sn phm PCR ca on ADN ny ( Knh 1 v 2, 3, 5). Knh 2 cho thy plasmid ca khun lc xanh khng mang on ADN ngoi lai, v ch c mt bng duy nht c kch thc vo khong 3,9kb l trng lng phn t ca plasmid.

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