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#1 Re: rf-cloning troubles » Is phosphorilation needed? » 2014-10-15 14:03:20

Thank you very much for the very quick reply!
i will probably set up the experiment with both methods
2nd PCR with EMP--> phosphorilation--> ligation --> DpnI
2nd PCR --> DpnI

I will see if i get major differences.

Thanks again


#2 rf-cloning troubles » Is phosphorilation needed? » 2014-10-15 13:14:19

Replies: 18

Hi everyone
I have a very simple question. I was thinking of using the exponential Megaprimer PCR described by A. Ulrich 2012. In this method a Phosphorylation step is mentioned as well a ligation before the treatment with DpnI. On other publications ( Methods Mol Biol. 2014;1116:73-87) there is no mention of such step and right after the 2nd PCR DpnI is added to the mix and used for the E. coli transformation.
At a first glace in made sense to have such steps included but the other article made me wonder. Is it actually needed? If not how does the "linearize" vector get ligated in the cells since the ends are not phosphorilated?

p.s. there is no mention of the need of phosphorilated primers

Thanks in advance for the clarification



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