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One Tube PCR Cloning Method
Content sponsored by New England Biolabs
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!
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hello Dr Oswald
thanks for the site it’s really helpfull!
i’d like to know if this protocol is convenient for blunt end cloning ( i use T4 DNA pol to blunt my vector and insert as well) and what about the buffers ?