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Cloning into a pGEX lacking of one RE consensus


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#1 anara

anara

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Posted 16 September 2010 - 10:08 AM

Hello everyone.
I'm a new registred on this forum but I've been reading it for a while now and every time I found out the resolutions to my problems or doubts so thank you all!!!

Now I have another question to make.
I have an insert which was designed for another experiment and plasmid which has BamHI sequence at 5' and XbaI at 3'.
I would like to clone it also into a pGEX with GST.
I dont have the pGEX with just GST, but one which already has an insert cloned with BamHI (5', beginning of the insert) and EcoRI (3' at the end).
If I cut out the insert I don't want with BamHI and EcoRI, then can I ligate mine by BamHI side and "adjust" the XbaI side with klenow reaction?
Or do you know any other way to make it?

thank you

#2 wincel

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Posted 24 September 2010 - 02:21 PM

Do a PCR on your plasmid with the wanted insert but have the restriction sites which exist in the target backbone pGEX at the 5' primer end. So you can replace the XbaI site with the EcoRI site by PCR primer.




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