分子生物學(xué)實(shí)驗(yàn)experimentsofmolecularbiology(參考版)
2024-10-16 11:18本頁面
【正文】 C for 20 minutes. ? 14) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes. ? 15) Wash the RNA once in 70% ethanol and vacuum dry. ? 16) Dissolve RNA in appropriate buffer . DEPCtreated, sterile TE, pH 8 or % SDS if no enzymic manipulation of the RNA is needed. SDS is an inhibitor of ribonucleases. ? RNA quality can be assessed by electrophoresis under denaturing conditions using agarose/formaldehyde gels and the MOPS buffer system. 。C for 20 minutes. ? 9) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes. ? 10) Wash the RNA once in 70% ethanol and vacuum dry. ? 11) Redissolve in 200ul % SDS at 65176。C, in solution D (500ul per 50mg tissue) with an eppendorf pestle homogenizer until a smooth, lysed, homogenous suspension is obtained. ? 2) Add 50ul 2M sodium acetate, and mix vigorously. ? 3) Add 500ul phenol and mix vigorously. ? 4) Add 100ul chloroform, mix vigorously and incubate on ice for 15 minutes. ? 5) Centrifuge mixture at 10,000g for 10 minutes in a microfuge at 4176。 ? You will need: ? Guanidine isothiocyanate (Sigma) 1M sodium citrate, pH 7 (DEPCtreated, autoclaved) Sarcosyl (Nalauryl sarcosine, Sigma) bmercaptoethanol (Sigma) 2M sodium acetate, pH4 (DEPCtreated, autoclaved) 3M sodium acetate, 100mM magnesium acetate, pH Absolute ethanol Propan2ol (isopropanol) 70% ethanol (made with DEPCtreated, autoclaved water) % SDS (made with sterile, DEPCtreated water) Solution D 4M guanidine isothiocyanate, 25mM sodium citrate, pH7, % sarcosyl, 100mM bmercaptoethanol. ? Solution D can be made and stored at 4176。 RNasin能與 RNase非共價(jià)結(jié)合從而抑制它們的活力 ， RNasin是 RNA酶的一種非競(jìng)爭(zhēng)性抑制劑 。H2O ?復(fù)合硅酸鹽 (Macaloid) ?肝素 ?氧釩基 — 核苷復(fù)合物 ?多胺 （ 2） 去除蛋白質(zhì)的物質(zhì)： ?蛋白質(zhì)變性劑：酚 、 氯仿；尿素 ?蛋白酶 K ?去污劑： SDS、 十二烷酰肌氨酸鈉 (sarkosyl)、脫氧膽酸鈉 (DOC)是陰離子去污劑 ?解偶劑 (胍類 )：鹽酸胍 、 異硫氰酸胍是一類強(qiáng)力的蛋白質(zhì)變性劑 ， 可溶解蛋白質(zhì) ， 并使蛋白質(zhì)二級(jí)
教學(xué)課件相關(guān)推薦
鄂ICP備17016276號(hào)-1