THC TP DI TRUYN LN

TO DNG V BIU HIN GENE E7 HPV18 TRONG T BO E.COLI THNG QUA VECTOR pET-28 (a) NI DUNG THC TP
NGY 1: Thu nhn gene E7 ca HPV 18 t t bo Hela. NGY 2: To plasmid pET28 ti t hp mang gene E7. NGY 3: Bin np plasmid ti t hp vo t bo E.coli DH5. NGY 4: Sng lc dng t bo mang plasmid ti t hp. NGY 5 : Tch chit v kim tra plasmid ti t hp c thu nhn bng phn ng ct gii hn NGY 6: Kim tra plasmid ti t hp c thu nhn bng phng php lai Southern Blot. NGY 7: Biu hin gene E7 trong t bo E.coli BL21 (DE3)

YU CU I VI SINH VIN c k quy trnh thc tp trc bui hc Mi nhm chun b 1 cun tp lm Lab book: ghi nhn li cc cng vic, thit k th nghim thc hin theo tng ngy; ngha cc thit k th nghim ; kt qu t c mi th nghim; phn tch kt qu (cui kha thc tp s thu li lab book chm im)

Mi nhm mang theo 1 laptop vo ngy 1 (13/12/2010)

- nh gi mn hc da trn: Bo co th nghim (lab book), Kt qu th nghim v Bi thi

cui kha

- Vng t 2 bui tr ln s cm thi. Trong thi gian hc nu mun xin ra ngoi trong 1
khong thi gian no phi lm n xin php trc.

- i tr qu 5 pht coi nh vng bui hc

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NGY 1: THU NHN GENE E7 CA HPV 18 T T BO HELA

I. Tch DNA t t bo HeLa.

Thu dch nui cy cha khong 2.106 t bo. Ly tm 5000rpm, 10 pht. Thu ta. Thm vo 500l Chelex. (Lu : vortex k Chelex trc khi s dng). Vortex k hn hp t bo v chelex trong 5 pht. mu 75oC trong 10 pht. trong ang tan 2 pht. Ly tm 13000 rpm trong 2 pht. Thu dch ni vo mt eppendorf 1,5 mi. Cn thn khng ht phn on c ht resin. Gi eppendorf trong . o OD vi bc sng 260nm v 280nm. II. PCR thu nhn gene E7 t DNA t bo HeLa bng cp mi E7fNde v E7rSal. Pha long mu PCR n nng 50 ng/l bng TE Thnh phn phn ng PCR (mi nhm t 2 phn ng): Master mix: Primer: ddH2O: Sn phm PCR (50 ng): Tng th tch: Nhm 1 t thm 1 phn ng nh sau: Master mix: Primer: ddH2O: plasmid pet28-E7HPV18: Tng th tch: 12,5 l 1l 10,5l 1 l 25l 12,5l. 1 l 10,5l. 1 l. 25 l

 Nhm 2 t thm 1 phn ng nh sau: Master mix: Primer: ddH2O: Tng th tch: Lun trnh nhit ca phn ng PCR
950C 940C 720C 5pht 30 giy 500C 30giy 1 2(lp li 39 ln) 3 720C

12,5 l 1l 11,5l 25l

1 pht

6pht

Trn 2 sn phm PCR li trong mt eppendorf 1,5ml in di 5l sn phm PCR trn gel Agarose 1% vi hiu in th: 80V trong 30 pht. HI: ngha cc phn ng nhm 1 v 2 lm thm?

III.

Tinh sch sn phn PCR.

Cho 260 l TE 1X vo sn phm PCR Cho 300 l phenol:chloroform. Vortex k trong 5 pht. Ly tm 13.000 rpm trong 10 pht 4oC. Thu ht dch ni pha trn vo mt eppendorf 1,5ml mi. Cho thm 1V chloroform, vortex k trong 2 pht. Ly tm 13.000 rpm trong 10 pht 4oC. Thu ht dch ni pha trn vo mt eppendorf 1,5ml mi. Thm 1/10V Sodium acetat 10X, 2V Ethanol tuyt i lnh, o eppendorf 4-5 ln.
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 -20oC trong 2 gi Ly tm 13.000 rpm trong 20 pht 4oC. Loi b dch ni. Nh nhng thm 1ml Ethanol 70% lnh. Khng c vortex. Ly tm 13.000 rpm trong 10 pht 4oC. Ha ta trong 20l TE 1X. o OD bc song 280nm v 260 nm. in di trn gel Agarose 1% vi hiu in th: 80V trong 30 pht.

NGY 2: TO PLASMID pET28 TI T HP MANG GENE E7.

I. X l plasmid pET28 (a) v sn phm PCR gene E7

Thnh phn phn ng ct: Vector pET-28a(+) /PCR-E7 Buffer D BSA 10X SalI(10u/l) NdeI(10u/l) dH2O Tng th tch phn ng ct 370C trong 3 gi. II. Tinh sch sn phm ct. 3,0 l (600 ng) 4,0l 4,0l 1,0l 1,0l 25,0l 40,0l

Cho 260 l TE 1X vo mi eppendorf cha sn phm ct Cho 300l phenol:chloroform. Vortex k trong 5 pht. Ly tm 13.000 rpm trong 10 pht 4oC. Thu ht dch ni pha trn vo mt eppendorf 1,5ml mi. Cho thm 1V chloroform, vortex k trong 2 pht. Ly tm 13.000 rpm trong 10 pht 4oC. Thu ht dch ni pha trn vo mt eppendorf 1,5ml mi (khong 200l) . Thm 1/10V Sodium acetat 10X, 2V Ethanol tuyt i lnh, o eppendorf 4-5 ln. -20oC trong 2 gi. Ly tm 13.000 rpm trong 20 pht 4oC. Loi b dch ni. Nh nhng thm 1ml Ethanol 70% lnh. Khng c vortex. Ly tm 13.000 rpm trong 10 pht 4oC. Ha ta trong 20l TE 1X. o OD bc song 280nm v 260 nm. in di

III.

Phn ng ni.

Cng thc thit lp phn ng ni.

Cho bit: M (kb) of insert = 0,3 kb; M (kb) of vector = 5,4 kb t hai phn ng ni: 1) t l ni (insert:vector) = 3:1 v 2) t ni (insert:vector) = 0:1 Thnh phn phn ng ni. 3:1 Sp PCR Plasmid pET28 (100-200ng/l) Buffer T4 DNA ligase10X T4 DNA ligase ddH2O Tng th tch 16oC qua m. 3l 1l 1l 1l 4l 10l T ni 0l 1l 1l 1l 7l 10l

NGY 3: BIN NP PLASMID TI T HP (pET/E7) VO T BO E.coli DH5.

I. X l sn phm ni bng enzyme BamHI.

Bt hot ligase 65oC trong 10 pht. t phn ng ct vi EcoRI: Sn phm ni Buffer H 10X BSA 10X EcoRI (10u/l) ddH2O Tng th tch 37oC trong 2 gi. HI: ngha ca bc x l ny? 10 l 2,0l 2,0l 0,5l 5,5l 20,0l

II.

To t bo kh np (PTN chun b) Hot ha 5 ml t bo E. coliDH5 trong mi trng LB, qua m. Nui 1 ml t bo E. coliDH5 trong 50 ml mi trng LB, lc n nhit 370C cho n khi dch t bo t OD t 0,3 -0,6. Ly tm 5.000 vng/pht, 5 pht 40C thu sinh khi. Ra sinh khi bng dung dch 25 ml CaCl2 100mM, trong 30 pht. Ly tm 5.000 vng/pht, 5 pht, 40C thu sinh khi. Huyn ph li trong 5ml dung dch CaCl2 100mM B sung Glycerol n nng cui l 12%. Aliquot thnh 5 eppendorf gi -80oC.

III.

Bin np

Mi nhm lm 5 phn ng bin np: A T bo kh 100L np Sn phm ni pET28 nguyn 3:1 B 100L T ni C 100L 1l D 100L E 100L -

Cho sn phm ni vo eppendorf c sn 100 l t bo kh np. trn 30 pht. Heat shock 420C trong 90 giy. trn nc ang tan trong 2 pht. B sung 1ml mi trng LB vo mi eppendorf, 37oC, trong 1 gi. Ly tm 10.000 vng/ pht trong 2 pht b dch ni. Cn li phn ta v 1 t dch ni, dng pipette huyn ph v chuyn ln a tri. Tri cc eppendorf A,B,C,D ln a LB c b sung Kanamycin(30g/ml), eppendorf E tri trn a LB, qua m nhit 370C. HI: ngha ca 5 phn ng bin np thc hin?

NGY 4: SNG LC DNG T BO MANG PLASMID pET/E7

IV. Sng lc khun lc bng phng php PCR vi cp mi T7 promoter v E7r-Sal

Quan st v ghi nhn, gii thch kt qu trn cc a tri A, B, C, D, E Mi nhm chn 5 khun lc trn a A v 1 khun lc trn a C thc hin 6 phn ng PCR Thnh phn phn ng PCR: Master mix: Primer: ddH2O: 12,5 l 1l 11,5l

Mt t sinh khi khun lc Tng th tch: Nhm 3 t thm 1 phn ng nh sau: Master mix: Primer: ddH2O: 12,5 l 1l 11,5l 25l

Mt t sinh khi khun lc E.coli DH5 mang plasmid pet28-E7HPV18 Tng th tch: Nhm 4 t thm 1 phn ng nh sau: Master mix: Primer: ddH2O: Tng th tch: 12,5 l 1l 11,5l 25l 25l

Lun trnh nhit.

950C 940C 720C 5pht 30giy 1pht 500C 30giy 1 2(lp li 39 ln) 3 6pht 720C

in di trn gel aragose 2%. Chn 1 khun lc cho kt qu PCR dng tnh vi insert, hot ha khun lc qua m trong 5ml mi trng LB-Kanamycin (30g/ml). Hi: ngha ca cc phn ng nhm 3 v 4 lm thm?

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NGY 5: TCH CHIT V KIM TRA PLASMID TI T HP BNG PHN NG CT GII HN

I. Tch plasmid ti t hp.

Chuyn dch nui cy vi khun qua m sang falcon 15ml Ly tm thu sinh khi 5ml dch vi khun nui qua m 5000 rpm trong 10 pht. Thm 200l dung dch 1. Trn u bng vortex. Chuyn qua 1 eppendorf 1.5ml mi. Thm 400l dung dch 2. o ng 4-5 ln. trn ang tan khng qu 5 pht. Thm 300l dung dch 3. o ng 10 ln. trn 15 pht. Li tm 13000 rpm trong 15 pht. Thu dch ni (khong 500l) vo mt eppendorf mi. Thm 50l Sodium acetat (1/10V), 1ml Ethanol tuyt i lnh o nh ng 2-3 ln -20oC trong 2 gi. Ly tm 13.000 rpm trong 20 pht 4oC. Loi b dch ni. Nh nhng thm 1ml Ethanol 70% lnh. Khng c vortex. Ly tm 13.000 rpm trong 10 pht 4oC. Phi kh ta 65oC Ha ta trong 20l TE 1X. B sung 2l RNAse (20mg/ml), 37oC trong 30 in di trn gel Agarose 1% trong 30 pht vi dng in mt chiu 80V. o OD

II.

Ct plasmid bng hai enzyme NdeI v SalI.

Thnh phn phn ng ct: Plasmid ti t hp Buffer D BSA 10X SalI(10u/l) NdeI(10u/l) dH2O Tng th tch 37oC trong 2 gi
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3,0 l (600 ng) 2,0l 2,0l 0,5l 0,5l 12,0l 20,0l

in di ton b 20l phn ng ct v 3l plasmid cha ct trn gel Agarose 1% trong 30 pht vi dng in mt chiu 80V.

III. -

Bin np vo E.coli BL21 (DE3) Chn 1 plasmid ti t hp c mang insert t kt qu ct gii hn mc II Bin np vo E.coli BL21 (DE3): Ht 1 l plasmid cho vo 100 l E.coli kh np. Quy trnh bin np ging quy trnh bin np vo t bo E.coli DH5 Mi nhm lm 3 phn ng bin np: A T bo kh 100L np Plasmid ti t 1l hp B 100L C 100L -

Tri eppendorf A,B ln a LB c b sung Kanamycin(30g/ml), eppendorf C tri trn a LB, qua m nhit 370C

Hi: ngha ca 3 phn ng bin np thc hin?

IV.

Lai Southern blot 1. Bien tnh

Sau ien di mc II, gel agarose se c bien tnh 30 phut trong dung dch NaCl 1M NaOH 0.5M. Sau o se c trung hoa 2 lan, moi lan 15 phut, bang dung dch NaCl 1.5M Tris HCl 0.5M pH 7.4. 2. Chuan b mang lai

DNA sau bien tnh se c chuyen tham len mang lai nylon (Hybond N+ - Amersham) trong SSC 20X , chuyen tham qua m. Mang lai co kch thc phu hp vi mieng gel mang DNA, c cat goc thang va anh dau mat chuyen nucleic acid len.

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NGY 6: KIM TRA PLASMID TI T HP BNG PHNG PHP LAI SOUTHERN BLOT
I. Th nghiem Southern Blot (tiep theo) 3. Tien lai

DNA tren mang lai sau khi chuyen tham se c co nh bang UV cross trong 10 phut. Mang lai c em tien lai 2 gi 420 C trong dung dch tin lai (SSC 6X, Denhardts solution 5X, SDS 0,5%, formamide 50%) co bo sung 50 g/ml DNA tinh trung ca hoi 4. Lai

Bien tnh mau do a anh dau vi Biotin-11-dUTP: un 1000C 5, lam lanh 5 Thc hien lai 5 gi nhiet o 420 C, trong dung dch lai co them mau do a c bien tnh. 5. Phat hien

Sau khi lai, ra mang lai 2 lan 10 phut/lan nhiet o phong vi dung dch SSC 2X SDS 0,1% Ra tiep 2 lan, 20 phut/lan, 650 C vi dung dch SSC 0,1X SDS 0,1% . Nhung mang lai trong dung dch em blocking/washing trong 5 phut. Khoa mang bang dung dch blocking trong 30 phut nhiet phong U mang lai vi dung dch Streptavidin cong hp vi Alkaline phosphatase(AP) trong 30 phut. Ra mang 2 lan, moi lan 15 phut, nhiet o phong vi dung dch em blocking/washing. U mang lai vi dung dch phat hien (detection buffer) trong 10 phut. Cho mang lai vao dung dch em co cha c chat NBT (Nitroblue tetrazolium) BCIP (5 bromo 4 cloro 3 indolyl phosphate) e trong toi, sau 15 30 phut xem ket qua hien mau. Khi cng o tn hieu at mc yeu cau, dng phan ng bang nc cat hai lan.

II.

Hot ha chng E.coli BL21 (DE3) mang plasmid ti t hp. Quan st, ghi nhn v gii thch kt qu cc a bin np ngy 5
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 Chun b mt ng nghim cha 5ml mi trng LB lng b sung Kanamycin (30g/ml) Dng u tp vng ly mt khun lc E.coli BL21 (DE3) mang plasmid ti t hp thu c trn a LB-Kanamycin (30g/ml) vo ngy 5 cho vo ng nghim. Nui cy lc 37oC qua m. Nhm 5 hot ha thm 1 ng t 1 khun lc E.coli BL21 (DE3) mang plasmid pET28 nguyn.

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NGY 7: BIU HIN GENE E7 TRONG T BO E.coli BL21(DE3)

I. Cm ng biu hin gene E7 bng IPTG

Chun b 3 ng nghim/nhm c 5ml mi trng LB lng c b sung Kanamycin (30g/ml). B sung 400l dch vi khun mang plasmid ti t hp/ng Nui cy lc 37oC cho n khi OD600 ca dch vi khun t trong khong 0,6-1,0. B sung IPTG 100mM vo mi trng nui cy theo bng sau: NG A IPTG 100mM 54 L NG B 54 L NG C 0 L

Nhm 6 lm thm 2 ng hot ha plasmid pET28 nguyn nh sau: NG D IPTG 100mM 54 L NG E 0 L

Nui cy lc trong 4 gi 37oC. HI: nng IPTG s dng cm ng biu hin l bao nhiu? ngha ca thit k th nghim cm ng biu hin? II. in di SDS-PAGE

Chuyn dch nui cy vo falcon 15ml. Ly tm thu sinh khi 5000rpm trong 10 pht. Ha sinh khi bng dung dch NaCl (50mM)/EDTA (1mM) pH8.0 theo bng sau: NG A NaCl/EDTA 500 L NG B 250 L NG C 500 L NG D NG E (nhm 7 lm) (nhm 8 lm) 500 L 500 L

Chuyn ht dch sinh khi ha tan sang 1 eppendorf 1.5ml em ng B i sonicate 10s, 5 ln. Cc ng khc gi trn Ly tm ng B 13.000 rpm, 20. Chuyn dch ni sang 1 eppendorf 1.5ml mi (B1). Phn cn ha li trong 250 L NaCl/EDTA (B2)
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 Ly 35l dch mi ng A, B1, B2, C, D, E v 7l dung dch np mu 6X. Trn u un 100oC trong 10 pht. Spin down. Np mu vo ging. in di dng in 100V trong 30 pht. Sau , in di tip tc vi dng in 120V cho n khi vch mu i ra khi bn gel. Nhum gel phn tch vi thuc nhum Coomasive Blue trong 30 pht. Gii nhum vi dung dch gii nhum cho n khi bn gel trong sut. Phn tch kt qu biu hin ca cc ng A, B1, B2, C, D, E

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PH LC
I. S cu to plasmid pET28a

II.

M s gene E7HPV18 trn NCBI: AY262282

III.

Mt s ha cht s dng A. Ha cht dng trong nui cy v cm ng vi khun

Mi trng Luria-Bertan (LB) (pH 7,0) Trypton 10 g

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Cao nm men NaCl Mi trng LB thch: LB + agar 15% (w/v) Mi trng LB-Kan: LB + Kanamycin (30 g/ml). Ha cht dung trong biu hin gene mc tiu

5g 10 g

Dung dch IPTG (isopropylthio--D-galactoside) 100mM (gi -200C) B. Ha cht in di Ha cht in di gel agarose TAE 50X Tris-HCl Acid acetic m c EDTA 0,5M pH8 Ha cht in di gel polyacrylamide. Gel polyacrylamide nng 12% c thnh phn nh sau: Gel phn tch (separating gel) dH2O Tris-HCl 1,5M pH 8.8 30% acrylamide: 0,8% bisacrylamide SDS 10% APS 10% TEMED Gel gom (stacking gel) dH2O Tris-HCl 1.5M pH 6.8 30% acrylamide: 0,8% bisacrylamide SDS 10% 2,8 ml 0,5 ml 0,66 ml 0,04 ml
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242g/l 57,1ml/l 100ml/l

3,3 ml 2,5 ml 4,0 ml 0,01 ml 0,01 ml 15l

APS 10% TEMED Dung dch np mu 6X (pH=6,8) Glycerol SDS DTT Tris-HCl pH 6,8 Bromophenol blue -

0,04 ml 10l

30% 10% (w/v) 0.6 M 0,5 M 0,012% (w/v)

Dung dch in di SDS-PAGE 1X (gi nhit phng) Tris-HCl Glycine SDS 25 mM 200 mM 0,1% (w/v)

Dung dch nhum gel (gi nhit phng) Coomasive brilliant blue Ethanol tuyt i Glacial acetic dH2O Dung dch gii nhum Glacial acetic Ethanol tuyt i dH2O 100 ml 200 ml 800 ml 0,625 g 112,5 g 25,0 ml 112,5ml

C. Ha cht trong tch chit plasmid Dung dch I : Glusoce Tris-HCl (pH8,0) 50 mM 25 mM
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EDTA (pH 8,0)

10 mM

- Dung dch II (pha trc khi dng)

NaOH SDS - Dung dch III: pH=5,0 Potassium acetate Glacial acetic - RNAse 20mg/ml (gi -200C) - Ethanol tuyt i lnh - Ethanol 70% - Dung dch TE 1X pH=8,0 (Tris-HCl 10,0 mM, EDTA 1,0 mM) D. Ha cht dng cho lai Southern Blot Denhardts solution 100X - 2% (w/v) BSA - 2% (w/v) Ficoll - 2% (w/v) PVP (polyvinylpyrolidone) SSC 20X pH 7,0 - NaCl 3M - Sodium citratre 0,3M E. CHNG VI KHUN: 3,0 M 0,2 M 0,2N 1%(w/v)

E.coli DH5: F, 80dlacZM15, (lacZYA-argF)U169, deoR, recA1, endA1, hsdR17 (rK, mK+), phoA, supE44, , thi-1, gyrA96, relA1

E.coli BL21 (DE3): F ompT gal dcm lon hsdSB(rB- mB-) (DE3 [lacI lacUV5-T7 gene 1

ind1 sam7 nin5])

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F. THANG CHUN Thang chuan 1 kb DNA: Thang chun unstained protein

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