Long PCR on a cosmid vector - Help needed to run PCR (Dec/08/2006 )
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.
Be careful, it is more likely introduce mutants in such long fragment by PCR amplificaiton and is hardly find out.
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.
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.