Plasmid Cut & Paste with Quikchange |
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Nick Oswald |
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I recently had a problem where I needed to cut out an expression cassette (a promoter coupled to a coding sequence) from one plasmid vector and paste it into another containing an expression cassette so I could get tandem expression of the two genes. The recipient vector had no suitable restriction sites for performing this operation so I was faced with the fairly lengthly task of introducing two new restriction sites on the recipient vector using quikchange, then amplifying the expression cassette and sub-cloning into the recipient. Not much fun.
Then after browsing through Pubmed, Geiser et al 
(Biotechniques 31:88-92 July 2001) came to my rescue with a modified quikchange method that allows this operation to be performed in two PCR steps. The technique involves using primers that allow the amplification of the fragment to be “cut out” and introduce 20-30 nucleotides that are homologous to the target integration site on the recipient plasmid. The PCR product is then used as primers in the second PCR step in which the whole recipient plasmid is amplified, resulting in the “pasting in” of the fragment to make the new tandem expression vector.
The authors report that fragments of up to 1.1 kb could be pasted… I managed to paste a 1.6 kb fragment. A word of advice: the PCR yield is very low in the second step so be sure to use ultra-competent cells as the authors advise. I performed a 75 microlitre reaction, concentrated it down to 10 microlitres using ethanol precipitation and transformed 3 microlitres into ultracompetent (1×107 cfu/microgram) E.coli using electroporation. From this I obtained around 100 colonies, 9/10 of which had the insertion.
If you have used this technique or an alternative approach, or if you have any questions about it, please leave a comment below. If you enjoyed this post and would like to receive more tech tips in the future, please subscribe.
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Gardo
Hi Nick, have you tried CPEC? It’s a method for cloning developed last year by Duke’s iGEM team. Basically, they have their primers tail complement the final vector 5′ and 3′ end sequences. Pretty much like the method you describe here. After the PCR fragment is amplified it is mixed in a 1:1 ratio with the vector and a 1-15 cycles of denature/annealing/extension are performed and the primer tails now work as new primers to complete your desired plasmid. So far I’ve tried it and got 8 fold more colonies over my vector alone control plate. I haven’t run my digests yet but once I do it I’ll post how it worked. If it works it’s going to be definitely favorite method from now on because it’s such a breeze to perform. Here’s the paper:
http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0006441