发信人: zzzzh (happy), 信区: Biology
标 题: Re: the way to do itRe: PCR产物酶切一问
发信站: Unknown Space - 未名空间 (Wed Sep 10 13:56:44 2003), 站内信件

搞个TOPO-TA载体前4步10min之内完成
【 在 comeandgo (春困秋乏夏打盹) 的大作中提到: 】
: digestion of PCR product can be very tricky, what i do is following:
: 1 purify your PCR product with qiagen kit
: 2 use T4 PNK phosphorylate your PCR product's blunt end
: 3 ligate your blunt PCR fragment into any vector cutted with blunt end enzyme
: (like EcoRV)
: 4 transformation and mini prep plasmid
: 5 then use hindIII and EcoRI cut the fragment out of vector
: 6 purify your hindIII-EcoRI fragment with kit and insert it into the vector
: you want to use
: this will add one more subcloning step but i am sure it will work
: 【 在 comeandgo (春困秋乏夏打盹) 的大作中提到: 】
: : 2 hours digestion won't have star activity


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※ 来源:．Unknown Space - 未名空间 mitbbs.com．[FROM: 152.16.]