Can Bacteria Repair Dephosphorylated Plasmids?
As per WYSIWYG's suggestion, I tried the digest and dephosphorylation again with SalI and ran a gel. The gel showed that SalI was less than ideal at cutting this plasmid.


As you can see, the most intense band is the bottom band, which is most likely supercoiled DNA, with the linear DNA I want appearing in the middle, and open circle DNA coming in third. I very carefully cut the middle band out and did a gel extraction. When I ligated this the control plate had 1 colony. Unfortunately, the experimental plates had between 2 and 6 colonies each. I am running minipreps on those now, will just have to see what I get.

This explains why the previous attempts produced so many colonies on the control plates, most of the DNA was uncut, so no amount of dephosphorylation would help it.

One thing that does bother me, is that my gel extractions seem like they have had really poor yields. I typically start a digest with 4 ug of plasmid. After phenol:chloroform extraction and isopropanol precipitation, I'll usually recover 2 - 3 or more ug of DNA. After gel extraction, I'm lucky to get 300 ng of DNA. To make things weirder, the liquid that comes off the spin column in the final elution step will gel up, like a low density agarose gel. The only explanation I can come up with is that residual agarose in the spin column is somehow forcing its way through the membrane and into the elution buffer. The goo clogs pipette tips and makes life difficult.