我的电泳图 除了有目标band 没有其他杂band 可是 背景却是一整条像是拖尾的东西 就连负控制都是如此 原以为是萃dna时没有将蛋白质处理好 於是在phenol那个步骤多萃几次（直到中间介面没有白色样出现） 在最後的步骤再加入1λRNase於37℃下作用10分钟 实在是想不出还有什麽方法可以改善了 请各位帮忙了看看是不是我有什麽是没注意到地 因为没有相簿可以放 如果要看图可能要给我你的msn或是mail 麻烦各位了 以下是我的protocol 1. Inoculate a 5 ml liquid culture of bacterial strain of interest in appropriate media. Grow until culture is saturated. 2. Centrifuge = 1.5 ml of the culture to collect pellet (2 min in microfuge).Remove supernatant. 3. Resuspend pellet in 567 ul TE buffer. Add 30 ul of 10% SDS and 3 ul of 20 mg/ml proteinase K (final [ ]'s of 0.5% and 100 ug/ml, respectively).Mix thoroughly and incubate 1 hr at 37℃. 4. Add 100 ul of 5 M NaCl and mix thoroughly. 5. Add 80 ul of CTAB/NaCl solution. Mix thoroughly and incubate 10 min at 65℃ . 6. Add an approximately equal volume (0.7 - 0.8 ml) chloroform/ iso-amyl alcohol (24:1), mix thoroughly, and centrifuge for 5 min in microfuge. (see white interface) 7. Remove aqueous, viscous supernatant to a fresh microfuge tube, leaving the interface behind. Add an equal volume of phenol/chloroform/iso-amyl alcohol (25:24:1), mix thoroughly, and centrifuge for 5 min in microfuge. 8. Transfer the supernatant to a fresh tube. Add 0.6 vol isopropanol to precipitate the nucleic acids (enough salt present).Rock tube back and forth until stringy white DNA ppt becomes clearly visible. At this point it may be possible to transfer the pellet to a fresh tube containing 70% EtOH with a micropipette. Alternatively, the ppt can be pelleted by briefly spinning at RT (room temperature). 9. Wash the DNA with 70% EtOH to remove residual CTAB and respin 5 min at RT to repellet it. Carefully remove the supernatant and briefly dry the pellet under vacuum. 10. Redissolve the pellet in 100 ul TE buffer. This may take up to 1 hr. Typically, 15 ul is sufficient for digestion and running out on an agarose gel in order to give a good signal during a Southern hybridization. --

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