Separation of double stranded DNA

[roniadam]

Separation of double stranded DNA

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Post by roniadam » Mon Apr 25, 2011 1:53 pm

Hi everyone:
I am interested to make a large insertion (780bp) in my vector. This 780 bp is a PCR product (megaprimer), I have used quikchange II mutagenesis kit but couldn't success?? any idea about how to success???
My thinking about the failing in this insertion is in the megaprimer ??? Any suggestion?

[JackBean]

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Post by JackBean » Mon Apr 25, 2011 5:39 pm

WHY do you have 780 bp primer? =-O

[roniadam]

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Post by roniadam » Tue Apr 26, 2011 4:37 am

Hi Jackbean

This is a piece of PCR product which is called megaprimer because I want to insert this piece into my vector.

[JackBean]

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Post by JackBean » Tue Apr 26, 2011 6:39 am

and how do you want to insert it? By PCR or by digestion&ligation?

[roniadam]

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Post by roniadam » Wed Apr 27, 2011 4:01 am

By mutagenesis kit (Quikchange II kit). It has been done before with another group and they succeeded in insertion but I was not. I emailed them but there was no reply that is why I posted this problem in this forum....

[JackBean]

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Post by JackBean » Wed Apr 27, 2011 11:38 am

OK, but how the kit works? By PCR, rigth? So why do you use such long megaprimer instead of using standard ~20mer?

[canalon]

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Post by canalon » Wed Apr 27, 2011 4:56 pm

JackBean, I suggest you Google the system/kit before you make more of a fool of yourself. It does not work like a PCR.
However I do not have a login to the Agilent website, and so could not explore the design of the primer. But that would be my best bet. That there is something going on with that specific primer. I imagine that the 780bp addition either do not anneal really well, or that it binds somewhere else in your plasmid. Maybe you can try conditions that are a bit more stringent (higher annealing temp, DMSO) if the kit allows that.
Other solution, try megaprimer PCR out of the kit (create 2 PCR products that have 20-25 bp in common and use them as primers for 5 cycles then pause and add the primers that are at both end of the fusion and go on for 25-30 cycles. You should get a fusion gene that you can easily clone by restriction ligation.

[JackBean]

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Post by JackBean » Wed Apr 27, 2011 6:22 pm

I don't know, what are you talking about, but I found this
http://www.chem.uky.edu/courses/che554/quikchange.pdf