H THNG IN DI (ELECTROPHORESIS SYSTEMS)

G.V.H.D: Nguyn Hong LcS.V.T.H: Ng Ngc NhH THNG IN DI(ELECTROPHORESIS SYSTEMS)

A. H thng in di DNA nm ngangNguyn l hot ng

II. Thnh phn cu to v c im k thut ca my in di Sub-cell GT AgaroseThnh phn cu to

2. c im k thut

III. Vn hnhChun b DNA gel:Cn mt lng agarose nht nh ph thuc vo yu cu nng vo th tch agarose gel.

Bng 1: Nng gel ph hp vi kch thc DNA cn phn tch.

Bng 2: Th tch gel yu cu.- Cho agarose vo trong bnh cha c th tch thch hp(e.g., 250 ml bnh nn, Wheaton bottle, etc.), thm mt lng m ph hp v khuy. - C th lm nng chy agarose bng nc si, bng a t, hay bng l vi sng. Ngi ta thng dng l vi sng nhiu hn, ci t thi gian khong 5 pht sau khong 30 giy, ri ly ra a nhit v khong 60 oC bng nc lnh trc khi gel. Ch : Lun mang cc dng c bo v v mc o blue trong phng th nghim khi chun b v gel.

2. c gel agarosec gel trn Sub-cell base:- t Sub-cell cho cn bng.- y trt cng gels ti cc rnh i din im cui ca b gel.- Cm lc c s rng thch hp to ra cc ging cha mu cn thit trn gel gn pha cathode.- Chun b nng v khi lng agarose thch hp trong 1x electrophoresis buffer. Khi agarose nng chy c lm lnh xung khong 50-60 C th chng ta gel vo gia.

Ch : Nu nhit gel ln hn 60 th c th lm plastic b gin ra v lm gim tui th ca Sub-cell base. V kt qu l b mt ca Sub-cell khng cn bng phng.- khong 20-40 pht cho gel ng c nhit phng.- Cn thn khi rt lc ra khi gel ng c v tho ri cng gel.

c gel trn UVTP tray.- t khay ln mt ci b v gi cn bng.- y cng gel theo cc rnh ti v tr i din cui b. Phi chc chn l b v rnh ca khay phi cn bng v u. Khi lng ca the gates th phi kn ngn chn s r r trong qu trnh c gel.- Cm lc c s rng thch hp to ra cc ging cha mu cn thit trn gel gn pha cathode.

- Chun b nng v khi lng agarose thch hp trong 1x electrophoresis buffer. Khi agarose nng chy c lm lnh xung khong 50-600c th chng ta gel vo gia- khong 20-40 pht cho gel ng c nhit phng.- Cn thn khi rt lc ra khi gel ng c v tho ri cng gel.

c gel trn UVTP tray s dng Gel-caster.

Lp rp khay UVTP gel c gel trn UVTP tray s dng Gel-caster.- Kim tra cn bng leveling bubble.- M kho v trt movable walk m phn cui ca gel caster bng cch xoay v nhn cht ln.- t phn cnh h ca UVTP tray st vi fix wall.- Trt MW n cnh ca UVTP tray.- ng kn khay, xoy cht cam bng cch xoay v n xung ng thi.

- Khi cht cam ri vo khe thch hp, tip tc xoay cht nu thy cn. Vic ny m bo ng kn cc cnh ca khay gel.- t lc vo khay thch hp ca khay. - gel 20-40 pht cho ng gel, ly lc ra.- M kho ca cht cam. Trt MW xa khay. Ly ra khi gel caster.- t khay ln b ca Sub-Cell, mu gn cc (-).- Cho gel chm trong 2-6 mm dung dch m.

3. Tin hnh in di- Mt s m thng c dng in di Nucleic acid trn agarose gel nh Tris-Acetate-EDTA (TAE) buffer or Tris-Borate-EDTA (TBE) buffer. - Chun b mu gel loading. - Cho thm loading dye cho ti khi t nng 1x, iu ny gip cho mu nng hn v chm xung y ging. - Load mu vo trong ging bng cch s dng mt pipets chun hoc Multichannel pipets.

- y np li cn thn, khng c lm xo trn mu. Np ch c gn theo mt hng xc nh ph hp .- Chng ta c th thay i dy ca gel, chiu di, nng gel v loi m c s dng trong din di.

4. Nhum v quan st DNA.

Quy trnh nhum EtBr:- Ly mt lng EtBr thch hp ( 0,5 g/ml) nhum t 15-30 pht. - Lm long thuc nhum bng cch ngm trong nc (15-30 pht) vi th tch bng th tch bng vi th tch thuc nhum dng.- Ra nh thuc nhum cn d trn gel bng nc trong thi gian ngn.- t gel ln trn UV transilluminator quan st v phn tch bc sng 302 nm.- Dng thit b chuyn dng chp v quan st hnh nh DNA.B. H thng in di protein thng ngNguyn l hot ng: tng t h thng in di DNA nm ngang.Cu to: in di SDS-PAGEH thng Mini-PROTEAN Tetra Cell

Hnh: cu to bung in di

Hnh: gi gelIII. Vn hnh1. Lp khun gel

2. gel: Polyacrylamide gel khng lin tc:

- t lc vo khun gel. nh du trn tm knh cch rng lc 1cm. Ly lc ra.- Chun b dung dch monomer. Kh kh dung dch di iu kin chn khng ti thiu 15 pht.- Thm APS v TEMED vo v gel n ch nh du. gel nh nhng trnh trn ln gel vi khng kh.- Ngay lp tc ph ln dung dch monomer bng nc hay t-amyl alcohol.

- Polymer ha trong vng 45pht n 1h. Ra b mt gel bng nc ct. Ch : thi im ny, gel phn gii c th c bo qun RT qua m.- Chun b dung dch monomer ca stacking gel. Trn tt c cc thnh phn ngoi tr APS v TEMED. Kh kh di iu kin chn khng ti thiu 15 pht.- Lm kh phn nh ca gel phn gii vi giy thm trc khi stacking gel.

- Thm APS v TEMED vo dung dch monomer, gel vo 2 tm knh. Tip tc cho n khi ti nh ca ShP.- Cho lc vo.- Cho polymer ha trong vng 45 pht n 1h.- Nh nhng ly lc ra v ra cc ging bng nc ct hay m chy.- Ra khung gel v gi vi nc ct v nc kh ion sau khi dng.

3. H thng in di v loading mua) Lp rp:

b) Loading mu:- m vo ln n mp ca tm knh bn ngoi.

- Load mu bn ngoi bung in di. Ch : load mu t t chng nm n nh di y ging. Cn thn khng lm thng ging.

Hnh: load mu vo ging4. t h thng in cc vo trong bung in di- t bung in di trn b mt phng, pha trc ca bung in di quay v pha ngi s dng, khi t ng hng, mu bn phi mu en bn tri.- t in cc vo, ch (+) vi mu - y m vo bung in di.5. Lp bung in di: y np bung in di. Sau khi kim tra xong, n np xung cho n khi np nm vng trn bung in di.

6. Ngun in: - 200V l in p c ngh thc hin SDS page7. Ly gel:- Sau khi hon thnh in di, tt ngun- M np v cn thn nng cc in cc ln. Loi b m chy.- M kha v ly gel cassettes ra.- Tch 2 tm knh mt cch nh nhng.- Ly gel ra bng cch ngm gel trong dung dch c nh, nh nhng khuy ng cho n khi gel tch khi tm knh.

C. IN DI TP TRUNG NG IN(isoelectric focusing IEF)Nguyn l hot ng

- Ngoi in di SDS, c th in di cc protein ty theo im ng in (isoelectric point, IEP) ca chng. Phng php ny c gi l in di tp trung ng in (isoelectric focusing, IEF). Trong dung dch m c pH bin thin lin tc (gradient pH), cc protein s phn ly n v tr tng thch vi im ng in ca mnh.

Hnh: nguyn l hot ng ca IEFII. Cu to: My PROTEAN IEF Cell.

1. Cc c tnh k thut:2. Bn ngoi:-Front panel display

3. Khay: c 2 loi khayKhay rehydration/equilibration.Khay focusing.

4. IPG strip (immobilized pH Gradient Strip)u im ca IPG strip:-Reproducibility: +c cung cp sn sng s dng +S thit lp Gradient nh s iu khin ca my tnh +Gradient pH khng thay i. +Gradient pH v chiu di gel c iu chnh cht ch cho bc phn tch chiu th 1.-D s dng: +C tm plastic nng khun acrylamide. +Bo qun trong t lnh cho n khi s dng +Strips d dng np vo nh ca gel bc in di chiu th 2 +C th np mt lng ln mu protein.-S lng: +C th chy mt ln 24 strips 7cm hay 12 strips 11 cm hay 17cm.

Broad range:pH 3-10, Narrow range:pH 3-6,5-8,7-10, 4-7, Micro range:3.9-5.1, 4.7-5.9, 5.5-6.7, 6.3-8.3III. Vn hnh.1. Rehydration- IPG strip c dehydrate do phi c rehydrate n t c dy ban u l 0,5 mm trc khi s dng. + Rehydration c mu:- np mu protein vo Readystrip IPG, ngi ta tin hnh rehydrate trong m c cha mu. - Chun b mu trong rehydration buffer. Nhn chung, s dng 5-100g i vi nhum Ag v ln n 1mg i vi Coomassive.

+ Rehydration khng c mu:- Mu c th c np vo IPG strip sau qu trnh rehydration trc khi chuyn sang focusing.

- IPG strip c dehydrate trong m rehydration m khng c mu v mu c np vo thng qua ci knh ca focusing tray.

a. Ci t cc thng s:a1. Rehydration: IEF Cell cung cp s la chn bao gm bc rehydration c iu nhit trong qu trnh chy in di ng in hoc lp chng trnh rehydration tch bit.+ Active rehydration:- Mu c cha trong m rehydration. - S dng in p 50V.- S dng khay Focusing.

a2. Peltier Platform temperature: PPT c th c iu khin trong sut qu trnh rehydration v bc in di tp trung ng in.Nhit c ci t dao ng t 10 n 25 C. a3. Cc thng s v nng lng: + Cc phng php tng in p: c 3 s la chn gia tng in p: nhanh(RAPID), thng (LINEAR), chm ( SLOW). + in p: in p ti a 10 000V. + Thi gian tp trung ng in: Thi gian c th c ci t ln n 99h. + Gii hn dng in: - Dng in ra ti a l 2.4mA. Gi tr in p ti a l 99A/strip. Trong chng trnh c ci t sn, gii hn dng in c ci t n 50 A ngn nga vic un nng qu hay lm chy IPG strip. + Hold step: - Hold step duy tr in p 500V cho n khi qu trnh chy c dng bi ngi s dng. b. in di tp trung ng in:- Khi IPG strip c rehydrate chng c th c tp trung khi c hoc khng c electrode wicks c lm t.- Lm t electrode wicks trong nc kh ion, thm nc tha i. t electrode wicks trc trip trn nh ca c cathode v anode trong focusing tray ngay trc khi focusing.

c. Ci t cc chng trnh:Chng trnh rehydration:- C 2 phng php rehydration khc nhau, passive v active. Khi s dng phong php active rehydration, IPG strip c t trong focusing tray, passive rehydration, IPG strip c th c t trong rehydration/ equilibration tray hay focusing tray.

Mn hnh biu din trn thit bChy chng trnh c sn:IEF Cell bao gm 3 phng php c sn. Mi phng php da vo cc cch m in p gia tng. Trong tt c cc phng php c sn th gi tr in p ti a da vo chiu di ca IPG strip (4000v/7cm, 8000v/11cm, 10000v/17cm). Mi phng php cung cp 1 cng in trng ti a l khong ~600V/cm ca IPG strip. Cng dng in gii hn l 50microA/IPG strip.

Hnh: Cc bc thit lp chng trnh c snChng trnh d tr:Chn mc Store method.Mn hnh s hin th nhng phng php c. Chn phng php mong mun.C th RUN, EDIT, DELETE phng php.Trong mc EDIT, c th thay i tn phng php, thay i tng bc trong phng php, thay i cc thng s.Sau khi EDIT c th save li hoc khng save m cho chy lun, c th i tn hoc thay th lun phng php c.

Hnh: Cc bc thit lp chng trnh d tr.Ci t phng php mi:

Sa cha phng php trong lc chy:2.2.in di chiu th hai:

Ngm IPG strip trong m cha SDS v dn strip ln nh ca 2nd D gel. C nh bng dung dch agarose 0.5-1% m bo mi lin kt tt gia gel v strip.Chy in di.

Hnh: kt qu in di 2 chiuTHE END