Protocol Online logo
Top : Forum Archives: : Molecular Biology

Long PCR on a cosmid vector - Help needed to run PCR (Dec/08/2006 )

Hello

I have cloned a DNA fragment of around 25 kb on a cosmid vector. I have the sequence of the flanking regions around the cloned fragment.
How can i PCR amplify the fragment out of the vector?
Which PCR conditions should I use then?

I need to use this fragment for subcloning later on:

Thanks for all your help.

-makosad05-

Be careful, it is more likely introduce mutants in such long fragment by PCR amplificaiton and is hardly find out.

-rye-

amplification of the whole vector in a big culture would be better as the probability of an error in PCR is bigger than in replication.

-fred_33-

I am not sure about RE site on cosmid vector,
I have digest lambda DNA clone with such size insertion by NotI (Not I sites are provided by lambda arms) and subclone it to Not I digested SK plasmid for amplfication.

-rye-