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Help for recombination problem in my big construct - (Oct/06/2005 )

sad.gif Hi, everyone, I am cloning a 6 kb insert into a pPNT vector (10 kb with my right arm). I am uasing DH5 alpha cell frrom invitrogen. I got almost 50 colonies but all of them are plasmid with deletion. Strange thing is it also creat an exta Bam H1 cutting sit! I tried Sure cell and Stle cells to avoid recombination but transformation effeciency is really low, nothing obtained so far. Who has experience in avoiding the recombination?




Are your DH5A chemically competent or electroporated?

With constructs over 10 kb, transformation efficiency drops substantially for chemically competent cells.

I really don't know what to do about the "extra" BamHI site.

You'd have to describe it in more detail in order for me to help you.

Have you sequenced it? etc...



are you absolutely positive that your vector is what you think it is?