One Tube PCR Cloning Method |
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Nick Oswald |
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I love shortcuts, and this one is very good. Chun-Ming Liu of Plant Research International has a number of molecular biology protocols on his website, but my favourite is his One Tube PCR Cloning Method. The protocol involves simply putting the vector, insert, restriction enzyme, ligase (and in his case polymerase for polishing the ends) into one tube, incubating at 16°C for 2 days and then transforming.
I have to say I was pretty sceptical, but I had a go using one of my own vectors and got pretty good results back. The background was higher than normal, but I certainly got the clones. I think that although this won’t work in all cases, it is a great method for routine cloning… thanks Chun-Ming!
Easier Gene Cloning With Positive Selection Vectors
An Easy Lysis / Homogenization Method for Any Sample
Sequence and Ligation Independent Cloning
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RHin
Hi, interesting method!
Does this work for double digestion too?
s_laub
I think this is based on TA cloning. The blunt cutter leaves blunt overhangs on pBS where Taq can add T-overhangs. The PCR product already has A-overhangs so these are ligated together.