2 replies to this topic

[OA17]

Hi!
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!

[akhshik]

hi,

what is the smallest and the biggest size of you fragments?

[OA17]

Hi!
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.