Bi 1 GII THIU TRANG THIT B, THAO TC C BN TRONG PHNG TH NGHIM SINH HC PHN T1. Mc ch Gip sinh vin bit cch s dng cc trang thit b thng dng v cc thao tc k thut c bn trong phng th nghim sinh hc phn t. 2. Yu cu-

Sinh vin bit cch s dng micropipette, cch vn hnh my li tm, t hp

v trng.-

Bit cch pha mt s dung dch ha cht thng dng dng trong th nghim.

3. Ni dung 3.1. Dng c dng trong th nghim-

Cc dng c thy tinh thng dng nh becher, ng nghim, pipette, a

petri, ng ong, erlen.-

ng tube: bng nha polyethylen hay polypropylen chu c nhit kh

trng (1atm, 121oC, 20 pht), nhit thp (-20oC) v mt s dung mi hu c, thng c dung tch 0,2ml; 0,5ml; 1,5ml; 2ml.0,2ml 1,5ml 2ml

Hnh 1.1 Cc loi ng tube thng dng1

iu chnh th tch Cn tay

Piston

-

Micropipette (pipetman): dng ht dung dch th tch nh, chnh xc

cao.Gi tr ngng

Cn gt u tip

Ca s hin th gi tr th tch

ng ht

u gn tip

Hnh 1.2. Micropipette

Lu khi s dng:

Khng pipette tip xc vi dung mi hu c, ho cht. Khng gn ngun nhit (n cn, ).

2

-

Tuyt i khng iu chnh di ngng th tch ti thiu v trn ngng th tch ti a. Khi s dng xong phi iu chnh pipette v th tch ti a. Cc u tip (cone): Cc micropipette lun s dng cc u tip. Cc u tip

ny thng ch c s dng mt ln v c nhiu loi tng ng vi nhiu loi micropipette khc nhau. Cc u tip ny c hp kh trng iu kin thng thng.

Hnh 1.3 Cc loi tip thng dng 3.2. Mt s thit b thng dng

My lc n nhit: c s dng cho cc phn ng cn nhit n nh (phn ng enzyme, lai,). My bao gm bung c nhit iu chnh c i km vi b phn lc.

T ht kh c: s dng trong cc th nghim vi ha cht bay hi c hay c mi kh chu nh phenol, chloroform, -mercaptoethanol

Ni hp t ng (autoclave): dng hp kh trng cc dng c v ho cht th nghim.

T cy v trng (laminar flow): c s dng khi cn thao tc trong iu kin v trng nh tch chit, pha ha cht T phi lun gi sch, b mt th

3

nghim c kh trng bng cn. Trc v sau khi s dng phi thanh trng bn trong t bng cch bt n UV trong t nht 15 pht.

T lnh: l t mt (4oC) hoc t lnh su (-20oC, -80oC ) dng gi cc sinh phm, ha cht v mu th nghim cn gi nhit lnh. i vi mu, ho cht bo qun t lnh su cn x l ng cch v chn vt cha ph hp trc khi bo qun.

My ly tm: dng phn tch v thu nhn cc phn t khc nhau trong mt dung dch. Nguyn tc: s phn tch cc phn t khc nhau trong mt dung dch c

thc hin da trn vn tc lng khc nhau ca chng di tc dng ca mt lc ly tm. Ty thuc vo c im dng phn tch (khi lng hay t trng ca cc phn t vt cht) m ngi ta chia lm 2 loi: ly tm phn on (ly tm vng) v ly tm ng t trng. * Ly tm phn on: dng phn tch cc phn t vt cht khc nhau da vo khi lng ca chng. Vn tc lng ca cc phn t ph thuc vo lc ly tm, khi lng, t trng v lc ma st ca cc phn t vi dung dch ly tm. phn tch thnh cng cc phn t c trong mt dung dch cn phi c lc ly tm v thi gian thch hp. * Ly tm ng t trng: phn tch cc phn t vt cht da vo t trng ca chng. Phng php ny rt hiu qu, cho cc phn on phn tch thun khit. ng ly tm cha mt ct dung dch c t trng tng dn t trn xung y ng to nn gradient t trng. Cc phn t s lng trong qu trnh ly tm v nm vng dung dch c t trng tng ng vi n. Saccharose v glycerol c dng khi phn tch cc bo quan, cesium chloride (CsCl) c dng phn tch cc protein v nucleic acid.

4

Hnh 1.4 My ly tm Ch : khi ly tm phi cc tube i xng nhau trn roto i vi s tube l s chn, nu s tube l phi thm mt tube cng loi v thm vo th tch nc bng vi th tch ca cc tube kia ri thc hin ly tm.

My vortex: gip lm ng nht ha mu hoc dung dch mt cch nhanh chng. My lun nhit (Thermocycler): l thit b gip thay i nhit ca hn hp dung dch phn ng PCR mt cch t ng theo chng trnh ci t trc ca ngi s dng.

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Hnh 1.5 My lun nhit 3.3. Chun b ha cht 3.3.1 Mt s nguyn tc pha ha cht

Ha cht c pha vi nc ct hai ln v kh trng iu kin thng thng. i vi mt s ha cht khng th hp kh trng, vic thanh trng c tin hnh vi u lc c ng knh l lc l 0,2m hoc 0,45m.

Khi ong, cn ha cht tuyt i khng ho cht tha tr li bnh cha ban u.

3.3.2. Tnh ton cc ha chta. Nng Mol: nng phn t gram theo th tch hay s mol cht tan trong mt lt

dung dch. c dng biu th nng mol ca mt dung dch. V d: pha ch 100ml dung dch 5M NaCl (58,456 g/mol) cn 5 mol/lt x 58,456 g/mol x 0,1 lt = 29,29 g NaCl trong 100 ml dung dch b. Phn trm Phn trm (w/v): s gram trong 100 ml dung dch Phn trm (v/v): th tch (ml) trong 100 ml dung dch

VD: to dung dch 0,7% agarose trong TBE buffer, cn 0,7 gram agarose v thm dung dch TBE n 100 ml.c. Dung dch m c: vi dung dch m ca enzyme c chun b di dng dung

dch m c nh 5X hoc 10X (m c gp 5 n 10 ln dung dch s dng), sau c pha long n nng cui (thng l 1X hoc 0,5X). 3.3.3. Chun b dung dch lm vic (working solutions) t dung dch m (concentrated stock solutions).

6

Mt vi dung dch m c s dng trong cc th nghim vi cng thnh phn c trong dung dch nhng vi nhng nng khc nhau, cn phi chun b nhng dung dch m c (stock solutions) ban u c th pha long khi cn. chun b 100 ml buffer TE (Tris 10 mM, EDTA 1mM) bao gm 1ml Tris 1M v 0,2 ml EDTA 0,5M v 98,8 ml nc ct. Cng thc tnh ton nh sau: C1 x V1 = C2 x V2 C1 : nng ban u ( hoc nng ca dung dch m) V1 : th tch ban u (th tch ca dung dch) C2 : nng cui ( hoc nng cn) V2 : th tch cui (th tch cn) 3.3.4. Mt s phng php c bn bo qun ha cht Cc dung dch thng c pha v bo qun di dng m c (dung dch m).

Dung dch m c pha long vi nc ct hai ln kh trng t nng cn cho mi ln s dng. Dung dch s dng c th bo qun nhit phng th nghim, nhit 4o

C hoc nhit lnh su -20 oC theo khuyn co cho tng loi ha cht. Dung

dch bo qun -20 oC thng c phn thnh nhng phn nh, mi ln s dng ch cn gii ng mt phn. 4. Thc hnh Sinh vin quan st cc thit b, dng c, cch vn hnh, thc tp vi

micropipette.-

Ch n cc n v o lng: th tch, nng v cch pha mt s dung

dch gc nh EDTA 0,5M; NaCl 3M; TE 5X; TBE 10X, dung dch li trch DNA, tnh lng ha cht cn thit cho phn ng PCR. 5. Kt qu t c7

- Sinh vin bit vn hnh cc thit b v s dng micropipette thun thc. - Sinh vin bit cch tnh ton v pha cc ha cht, dung dch thng dng.

8

Bi 2 LY TRCH DNA TNG S THC VT1. Mc ch: Gip sinh vin hiu phng php tch chit DNA thc vt. 2. Yu cu: Sinh vin nm vng v thc hin c cc bc tch chit DNA theo phng php CTAB v phng php dng dch trch SDS. 3. Ni dung 3.1 Gii thiu DNA l phn t c kch thc ln, do trong qu trnh thao tc cn trnh nhng tc nhn c hc hay ha hc qu mnh c th lm t gy phn t ny. Qu trnh tch chit DNA gm cc bc c bn nh sau: 1. Ph vch t bo, mng t bo v mng nhn (nghin mu, s dng cc cht hot ng b mt, proteinase). 2. Gii phng DNA s dng buffer tch chit bao gm cht hot ng b mt. 3. Lm bin tnh v loi b tp cht nh protein, cc sc t bng cch s dng chloroform hay hn hp phenol/chloroform. 4. Phc hi DNA bng cch kt ta 5. Ra v loi b cht ty v cc mui. 6. Thu hi DNA (7. S dng Rnase phn hy RNA) 3.2 Qui trnh li trch DNA tng s cy u xanh bng dch trch SDS

Dung dch ng nht mu (Homogenization Buffer) gm: Tris 200 mM (pH 8,0),

EDTA 25 mM, NaCl 250 mM, SDS 0,5%.- Bc 1: Cho mu l u xanh vo ci, b sung ni-t lng v nghin cho n khi

mu dng bt mn. Ngay sau , chuyn mu sang tube 1,5ml v cho 400 l dch ng nht mu vo v lc u.- Bc 2: B sung vo tube 400 l PCI (phenol/chloroform/isoamyl alcohol

(25:24:1)) v lc nh 1 pht, li tm vi vn tc 13.000 vng/5 pht, sau ht dch ni cho vo ng tube mi.9

- Bc 3: Cho vo tube 370 l PCI, lc nh 1 pht, sau em li tm 13.000

vng/5 pht. Ht dch ni chuyn qua tube mi. - Bc 4: B sung 340 l chloroform vo, lc nh 3-5 pht, li tm 13.000 vng/5 pht, ht dch ni chuyn qua tube mi. Thm 600 l ethanol 990, lc nh t 5-10 pht. -200C trong 30 pht. Sau li tm 13.000 vng/10 pht, b dch ni, thu phn ta. Phi tube nhit phng.- Bc 5: Thm vo kt ta DNA 30 l nc kh ion hp kh trng (khng cha

DNase) hoc 30 l TE 0,5X, vortex nh v bo qun 40C hoc -200C. 3.4 Ly trch DNA plasmid vi khun Gii thiu Qui trnh ly trch plasmid ny c dng ly trch DNA plasmid t huyn ph t bo vi khun v da trn qui trch ly trch kim tnh pht trin bi Birnboim v Doly (1979). Qui trnh ly trch da trn s khc bit v hnh thi gia DNA plasmid cc phn t nh, siu xon, v DNA nhim sc th - ln hn rt nhiu v t xon. S khc bit ny gip phn bit gia kt ta DNA nhim sc th, protein t bo vi plasmid v cc phn t RNA. iu kin kim, cc t bo c phn gii, c nucleic acid v protein u b bin tnh. Khi dung dch c trung ha bi Kali Acetate (CH3COOK), DNA nhim sc th v protein kt ta v cc phn t ny khng th phc hi li cu trc ban u (do kch thc ln). Cc plasmid hi tnh n nh v tn ti trong dung dch, tch hn ra khi DNA nhim sc th v protein. Vt liu Thit b - Ha Cht Vt liu, thit b ng 1,5 mL bnh tam gic My lc My ly tm My vortex Bn Ha cht Mi trng LB lng Dung dch I (lnh) Dung dch II Dung dch III (lnh) Isopropanol Nc (kh ion, tit trng)10

Dung dch I (bo qun 4oC) 50mM glucose (lc kh trng) 25mM Tris-HCl (pH 8.0) 10mM EDTA (pH 8.0)

Dung dch II 0.2 N NaOH (pha long t dung dch 10N khi s dng) 1% SDS

Dung dch III (bo qun 4oC) 5M potassium acetate (Kali acetate) Glacial acetic acid

Qui trnh ly trch DNA plasmid (Birnboim v Doly, 1979) Vi khun E.coli mang plasmid c nui cy trong mi trng LB lng (trypton 1%, Yeast extract 0.5%, NaCl 1%) 37oC, tc lc 200 rpm. Dch huyn ph vi khun sau 16 gi nui cy (nui qua m) c s dng ly trch plasmid theo qui trnh gm cc bc nh sau:

1.

Ly 1,5 mL dch khun cho vo tube 1,5 ml. Ly tm dch khun tc 10.000 rpm trong 1 pht. Loi b ht dch ni. Nh nhng p tube ln giy thm loi mi trng ra khi kt ta.

2. 3.

Thm 100 l dung dch I (c gi lnh), vortex k. Thm 200 l dung dch II. o tube trn u hn hp. Khng vortex bc ny. Gi tube trn .

4.

Thm 150 l dung dch III (c gi lnh). o tube trn u hn hp. Gi tube trn khong 1 pht.

11

5. 6. 7.

Ly tm 10.000 rpm trong 10 pht. Chuyn dch ni sang tube mi (1V). Thm 2V isopropanol, vortex nh; yn trong vng 20 30 pht -20oC. Ly tm 13.000 rpm/ 5 pht thu kt ta DNA. Loi dch ni pha trn. Thm 500 l ethanol 70%, ly tm 13.000 rpm/5 pht. Loi b dch ni v phi ng cho n khi khng cn dung dch no bn trong ng.

8.

Thm 30 40 l nc kh ion (khng cha Dnase) hoc TE. Vortex nh v bo qun -20oC.

BI 3

NH LNG DNA BNG K THUT O MT QUANG

1. Mc ch: Gip sinh vin hiu cch nh lng nucleic acid bng phng php o mt quang. 2. Yu cu: Sinh vin o lng mu v tnh nng nucleic acid 3. Ni dung 3.1. Nguyn tc Da vo mc hp th nh sng ca cc phn t vt cht trong mi trng lng ngi ta c th tnh c nng ca chng. Mi phn t c mt nh hp th (ni chng hp th nh sng mnh nht) mt di sng nht nh ty thuc vo cu trc ca chng. S hp th ny l do s tng tc gia cc proton vi cc electron ca vng purin v pyrimidin. nh hp th ca nucleotic l 260nm trong vng t ngoi. S hp th c tnh bng gi tr OD (Optical Density). Mt n v OD tng ng vi mt nng l: 50g/ml DNA si i 40g/ml DNA si n hay RNA 20g/ml oligonucleotide si n.

12

Hay c th tnh bng cng thc DNA ng/l = 62,9*OD 260- 36*OD280 T gi tr OD o c, ta suy ra c nng acid nucleic trong dung dch. Dung dch acid nucleic c xem l sch khi t l OD260/OD280 nm trong khong 1,8-2,0. Nu mu nhim protein hay phenol t l ny s thp hn v phng php ny khng chnh xc. 3.2.Phng php-

o OD260 ca dung dch DNA mu o OD280 ca dung dch DNA mu. Tnh t l OD260/OD280. un cch thy trong 20 pht dung dch DNA mu, lm lnh t ngt trong nc trong 10 pht. o OD260 ca dung dch bin tnh. Quan st gii thch kt qu v kt lun.

4. Kt qu t c Kt qu o OD

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BI 4 K THUT IN DI1. Mc ch Gip sinh vin hiu phng php thu nhn mu acid nucleic phn tch, nh tnh v nh lng DNA 2. Yu cu - Thc hin gel agarose 1%, np mu, thc hin in di v quan st gel di tia UV. - Xc nh hm lng dung dch acid nucleic bng phng php o OD.

4.1 in di acid nucleic Gii thiuQu trnh in di s dng gel agarose hay polyacrylamide thng c dng phn tch nucleic acid da vo kch thc. K thut in di c th c s dng trong phn tch hay phi hp vi cc k thut khc, c th nh tnh v nh lng. Nhng on DNA ln nh l nhim sc th c th c phn tch bng k thut in di ci bin pulsed field gel electrophoresis (PFGE), s dng s thay i chiu di chuyn ca DNA. K thut in di n gin nht v c s dng rng ri nht l k thut in di ngang. quan st DNA, cn phi nhum phn t ny, thng s dng ethidium bromide. Cht nhum ny bm vo DNA bng cch chn vo gia cc cp base i din nhau v to hunh quang cam/ khi c chiu tia UV. Cc cht nhum thay th khc vi nhy tng ng v t nguy nguy him hn, l SYBRGreen hay Gelstar. Gel agarose c th c s dng phn tch cc phn t ln hn khong 100 bp. Agarose l mt dng polymer c th c un ha tan, khun v to thnh nhiu ming gel vi kch thc khc nhau (thng l minigel c chiu di 10 cm). Cc ging (well) c nh hnh bng lc gel (comb) sau khi gel vo khun. Ming gel c t ngp chm trong dung dch in di v DNA c thm vo cc ging. Dng in i qua bn in di, theo chiu di gel lm cc phn t DNA tch in m di chuyn v pha14

cc dng. Agarose gel l mt ma trn gm cc l v cc phn t DNA c kch thc nh s di chuyn qua d dng hn cc phn t DNA c kch thc ln hn. Vi yu cu phn gii cao hn hay vic tch chit cc phn t DNA ngn hn nn s dng gel polyacrylamide.

Hnh 4.1 Ethidium bromide (a) ethidium bromide; (b) qu trnh chn vo phn t DNA ca ethidum bromide

Vt liu Thit b in di agarose gm: B cung cp ngun in Bn in di Khay, lc gel Gi gel (gel caster) Ha cht Dung dch m in di (TAE hoc TBE) Agarose Loading dye Cch chun b dung dch m15

1x Tris-Acetate-EDTA (TAE) (c pha long t dung dch stock c nng cao hn) gm Tris 40 mM (pH 7,6), acetic acid 20 mM, EDTA 1 mM. 50x TAE Stock (1 L). Ha tan trong 600 mL nc hp kh trng: 242 g Tris base (FW = 121); 57,1 ml glacial acetic acid; 100 ml EDTA 0,5 M (pH 8.0), thm nc vo n th tch cui cng 1L.

1x Tris-Boric Acid-EDTA (TBE) (c pha long t dung dch stock c nng cao hn) gm Tris 89 mM (pH 7,6), boric acid 89 mM, EDTA 2 mM 10x Stock (1 liter) Ha tan trong 600 mL nc hp kh trng: 108 g Tris base (FW = 121); 55 g boric acid (FW = 61.8) 40 ml 0.5 M EDTA (pH 8.0) thm nc vo n th tch cui cng 1L. DNA and RNA sample Loading Dye Dung dch m chy mu 10X gm glycerol 50%, bromophenol blue 0,25%, v xylene cyanole 0,25% trong m TAE 1X. Dung dch m chy mu thng c chun b vi th tch t 110 mL.

Hnh 4.2 H thng in di gel agarose (a) Ngun in; (b) bn in di (Mini-Sub Cell GT, Bio-rad)16

Khay gel

Gi gel vi cc thanh ngang c th di chuyn, thch hp nhiu c gel khc nhau.

Bn in di

Gi gel

Thnh c nh

Hnh 4.3 Cch chun b gel v t gel vo bn in di Cc bc thc hin Chun b gel 1. Xc nh lng agarose cn dng t c nng agarose v th tch mong mun. V d, pha 100 mL agarose 1% cn phi thm 1 gram agarose vo 100 mL buffer in di (0,5X hay 1X).

Bng 4.1 Cc nng gel dng trong phn tch DNA

Nng gel (%) 0,5 0,75 1,00 1,25 1,50 2-5

Kch thc DNA (kb) 1 30 0,8 -12 0,5 - 10 0,4 - 7 0,2 - 3 0,01 0,517

2. Thm lng Agarose thch hp vo bnh cha (v d nh bnh tam gic 250 mL, chai c np y 100 mL). Thm vo lng buffer in di thch hp (0,5 X hay 1 X) v lc phn tn agarose u trong buffer. Ghi ch: c th nh du mc cht lng trong bnh. Nu cht lng b bc hi, c th thm nc vo t th tch ban u.

3. Cho dung dch gel vo l vi sng v un si cho n khi cc ht agarose tan hon ton. Ngng microwave sau 30 giy v lc u s lm agarose tan nhanh hn Lu : lun mang gng tay, o blouse trong khi chun b v gel. Bnh cha agarose nng c th gy phng nu tip xc trc tip vi da. Ngoi ra, agarose nng chy c th vng ln v lm phng trong qu trnh lc.

4. Lm ngui agarose xung khong 60oC trc khi gel vo khay c mang lc

gel.

Lu : c th thm vo 0,5 g/mL ethidium bromide vo agarose nng chy.

Chun b bn in di5. Khi gel ngui v c li, khay gel c t vo bn in di, ngp trong dung dch

m in di. Lc gel c tho ra to nn cc ging rng dnh cho mu in di vo. Mu c bm vo ging bng micropipette (xem Chun b mu). C th thc hin in di vi cc loi dung dch m in di khc nhau nh Tris-AcetateEDTA (TAE) hay Tris-Borate-EDTA (TBE). Buffer TAE to s di chuyn trong in trng nhanh hn cho DNA mch thng v phn gii tt hn cho DNA siu xon;18

m TBE c kh nng m mnh hn cho qu trnh in di vi thi gian di hoc vi hiu in th cao hn. Chun b mu 6. Lng mu s dng ph thuc vo dng lc s dng ( dy v chiu di) v dy ca gel.7. Khi lng mu s dng chy gel c xc nh, thm loading dye vo vi nng

cui l 1X. Cht nhum ny c tc dng gi mu y ging in di v gip theo di s di chuyn ca mu trong qu trnh in di.8. Bm mu vo ging bng pipette.

Ghi ch: cc ging gel thng kh nhn bit. nhn thy cc ging r hn c th t tm giy hay bng keo mu en bn di bn in di, v tr cc ging. in di9. y np bn in di cn thn. Khng chm vo mu. Thng cc np bn in di

ch c th lp vo bn theo 1 hng da vo v tr nh du cc in cc.10. Ngun in cung cp thay i ty theo dy, chiu di v nng ca gel, dng

dung dch m in di s dng. Hiu in th t 20 110 V c s dng cho hu ht cc mc ch. 11. Nhum gel C 3 phng php c bn nhum v quan st nucleic acid khc nhau s dng ethidium bromide (EtBr): Thm 0.5 g/ml trc tip vo gel agarose Thm 0.4 g/ml trc tip vo mu v chy mu trn gel khng cha EtBr Nhum gel sau khi in di xong. Ngm gel trong dung dch EtBr.

19

Ghi ch: EtBr l cht gy ung th v cn phi thao tc ht sc cn thn. Lun mang gng tay, mt knh v o blouse. B dung dch EtBr s dng v gel ng cch. QUI TRNH NHUM ETHIDIUM BROMIDE SAU KHI IN DI1. t gel vo dung dch EtBr thch hp (0,5 g/ml), nhum 15 30 pht. S dng

dung dch nhum ngp ton b gel. 2. Ra gel 10 30 pht bng cch ngm gel trong nc. Ghi ch: Ethidium Bromide c th b loi khi DNA sau qu trnh ra. Qu trnh ra gel cng lm gim nhy ca qu trnh pht hin DNA. Tuy nhin, thi gian ra gel ngn cng to ra tn hiu nn cao. 3. Ra li gel bng nc loi EtBr cn bm trn gel. 4. t gel di tia UV quan st v phn tch DNA. Phc hp DNA/EtBr pht sng di tia UV bc sng 254, 302 hoc 366 nm.

20