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Cloning and PCR Problems - (Jun/30/2005 )

I just wanted to throw a couple of problems I have been having out there to see if anyone has any suggestions.

1. I have been trying to clone a 5 kb piece of genomic DNA into a targeting vector that is 12.5 kb that also contains a region of genomic DNA using a sticky end ligation with SacII. I have verified that I have complete digestion by visualizing and purifying the fragments on a gel. When I run the ligation and controls that contain vector only and insert only I get lots of colonies on the ligation plates and barely any colonies on the vector only control. The problem is that I am seeing a lot of colonies on the insert only control plates. I have screened colonies by restriction digest from the 5 kb and 12.5 kb ligation and get uncut DNA that will not cut with any enzyme that should cut it in several places. I have ruled out vector backbone contamination because I run-out the digested DNA very far before it is purified. It is almost like there is something in the genomic DNA that is allowing it to replicate and survive when transformed, although this makes no sense to me.

2. In a few recent PCR and restriction digests where there have been large quantities of DNA and the pieces themselves are large it appears that the DNA is not migrating out of the well and when observed under UV light, there is a bright band at the edge of the well. I have tried using 0.5% gels and have also tried boiling PCR samples prior to loading the gel. Has anyone had any experience with this and if so, how do you fix it? Thank you for your time.


For question 2.

I usually use the gel of 0.8% or 1%, and I didn't met that kind of problem before.