Problem: Cloning DNA shuffled band - (Feb/03/2012 )
I am currently doing DNA shuffling. The shuffling procedure itself is not a problem for me. I have optimised the initial PCR, DNAseI digestion, PCR without primers and reamplification with primers. Up to this point, everything seems to be going great. Afterwards I digest the shuffled DNA in he usual way and I try to clone it into my vector.
And here comes the problem: I am never able to get good colonies (tested by restriction digest).
My controls indicate that the restriction enzymes, ligase, etc, are working well. The electrocompetent cells are good, since I get plenty of colonies, but when I get to analyse them, they are all incorrect. Not a single good colony.
I have cloned a lot during my thesis, so I think I have enough experience. This one should be very easy. However, whenever I try to clone shuffled fragments, I have this problem.
Does anybody know wheher there is something special or something different to a normal cloning, that I should do in order to clone the shuffled fragment?
Thanks in advance!
what is the smallest and the biggest size of you fragments?
The insert is 2000 bp and the vector is 14 Kbp. I have cloned into this vector a hundred times. I'm very familiar to this kind of cloning and it should be very easy.