cloning problem - (Sep/08/2014 )

Hello everyone,

I am trying to replace a 40 bp insert of a 6,4 kbp vector, with another 40 bp sequence. This is flanked by restriction sites for NheI and HindIII, and we designed primers with hanging ends which after annealing would generate our insert. After digestion, I cut the backbone from the agarose gel and ligated with the annealed primers and after transformation I had some bacteria colonies. However, after isolating the plasmid DNA and running it on a 0,8% agarose gel I noticed that the plasmid runs at around 3 kbp. Do you have any idea what could have happened? I have more colonies to test, but I'm not sure if I will get different results.

Thanks a lot.

Anca

Check that the backbone doesn't have more than one cut site.

You might also want to check that you have either PNK treated your oligos or ordered them with 5' phosphates. (probably not the problem you are having, but perhaps an additional one).