RF-Cloning.org Forum

Anything and everything cloning: Go...

You are not logged in.

#1 2015-01-24 19:49:03

Registered: 2014-07-25
Posts: 11


What do you all think about the DpnI treatment for EMP cloning and/or RAM cloning?
In the RF cloning, I can see the point since its a linear amplification, but in EMP and RAM cloning you get an exponential amplification of the plasmid. I wonder of the parental plasmid needs to be destroyed in the end when you have a lot more amplified plasmid.
Any ideas on the necessity of the DpnI treatment/efficiency?


#2 2015-01-30 16:15:24

Steve Bond
Registered: 2014-01-23
Posts: 126

Re: DpnI

I think you should probably still do it, if for no other reason than to further reduce the chance of needing to screen false colonies. Do some back of the napkin calculations to estimate how many ng of parental plasmid you'd end up adding to you transformation after the PCRs are all finished.
With RAM cloning I think it would be more important, given that you're relying on recombination to form the final mature plasmid in vivo. I don't have any actual evidence to back this up, but I'd expect the parental plasmid to have an advantage here, even if it's at a much lower concentration than the linear DNA.


Board footer

Powered by FluxBB