c*******o
发帖数: 8869 | 1 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 sur | z***h
发帖数: 998 | 2 搞个TOPO-TA载体前4步10min之内完成
【在 c*******o 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 sur
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