IN DI GEL Phng php in di trong gel (keo) thng c s dng phn li (phn on) DNA, RNA, oligonucleotide, protein...

. sau c th dng gim bit (ng nh) v tinh ch cc cht . Vic phn li cc cht da trn ln cng nh hnh thi ca cc cht. Thng thng s dng gel agarose v gel polyacrylamide nhng gel agarose cho php phn li nucleic acid ln hn 1 kb, cn gel polyacrylamide thng dng phn li cc on nucleic acid nh hn 1 kb. Nguyn l ca vic in di DNA l khi trong in trng, do tch in m nn cc phn t DNA dch chuyn v pha anode v vi tc dch chuyn ca chng khc nhau ph thuc vo khi lng phn t. Cc on DNA cng ln th dch chuyn cng chm. Kt qu l t mt im chung (l hay "ging" ti mu) cc on DNA khc nhau dch chuyn v mt hng to thnh mt ln (lane), v trn ln c cc on DNA khc nhau phn b cc v tr (cc bng - band) khc nhau tng ng vi ln ca chng. Trong khi , cc phn t protein do tch in b mt khc nhau nu in di trong gel khng gy bin tnh th dch chuyn theo cc hng khc nhau vi tc khc nhau ph thuc c khi lng phn t ln in tch b mt, nhng nu in di trong gel sau khi x l bng SDS th do b mt protein tr nn tch in m ng nht nn chng dch chuyn v anode vi tc khc nhau hu nh ch ph thuc khi lng phn t. in di agarose 1. Ch tc gel agarose Di y gii thiu phng php ch gel agarose in di DNA. 1) Cho lng agarose (Seakem Agarose, Nusieve Agarose...) cn pha vo dung dch TAE 1 trong mt bnh tam gic theo bng di y c phn li nucleic acid thch hp. c dung dch TAE 1 cn cho thm 19 ln nc kh ion vo dung dch gc TAE 20. Nng agarose (%) ln DNA phn li (kb) 0,3 60 - 5 0,6 20 - 1 0,7 10 - 0,8 0,9 7 - 0,5 1,2 6 - 0,4 1,5 4 - 0,2 2,0 3 - 0,1 2) 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 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.

3) Cho vo chu bo n ~50 C 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).

Hnh 1: Cu to ca mt b in di nm ngang. Bn gel agarose c b sung ethidium bromide, sau khi in di c th quan st c cc bng DNA nu t chu trn ngun UV (cht liu ch y b thu qua i vi UV). Ch : Ethidium bromide l cht c gy ung th, v vy Cn 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. 4) Rt gel vo khun c chn hai u bng bng dn hoc bng kp, ci sn lc (comb) v t trn mt phng (xc nh bng thy bnh k, tc lig). 5) Ch cho n khi gel rn hon ton th cn thn tho lc ra khi gel. 2. in di agarose 1) 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. 2) 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). 3) Bng micropipet ht mu DNA nh vo l rng lc. 4) Bt in mt chiu thc hin in di. Cng dng in khong 50 - 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. 3. Kim tra bng DNA Hnh 2: Kt qu in di mt s on DNA trong gel agarose. Hai ln hai bn l du khi lng phn t (DNA molecular marker), mi bng (vt sng) ca mi ln ny cch nhau 100 base pair (bp), bng nh nht (di cng)

"nng" 100 bp, bng nng nht 2.000 bp (gia 1.000 bp v 2.000 bp khng c cc bng trung gian). c nh khi lng phn t ln DNA no th c th t mt thc thng ln n v dng ngang xem n tng ng vi bng no trong ln du khi lng phn t, trng hp khng trng kht th c th c lng. ngoi cng c th pht hin c cc bng DNA trong qu trnh in di cng nh sau khi in di. Kiu dng (pattern) cc bng ph thuc vo thnh phn DNA c trong mu cn in di. 1) Nu in di DNA trong gel c pha sn ethidium bromide th chiu tia t 2) 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. 3) 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.