Is OVER-dephosphorylation possible? - (Apr/27/2006 )

Hello all,

I have restricted an insect genome with Sau3A, selected fragments between 400 and 900 bp, with intent on making a library to probe for microsatellites. For this I need many hundreds of positive clones. I am having trouble with the number of positives, the usual ratio optimisation is not working.

I cut the plasmid myself (pUC19 / BamHI), and gels show complete digestion, then I dephosphorylated (Antarctic), but when I run ligation and transformation controls, the background of this linear plasmid is high (lots of blue colonies on the plate).

I will make the plasmid again, but can I leave the Antarctic phosphatase in the reaction for longer than an hour to get complete dephosphorylation?, someone told me this could be a problem.

Thanks,

Bevan

http://www.rhizobia.co.nz

hi
i don't think that this would be a problem. I don't have experienced over dephsphorylation. In fact my dephosphorylation is not complete. It seems that the quantity of material you start with is to much for the amount of antartic enzyme used. This may be adjusted too. I do dephosphorylation in 2 steps. First one at 37. Add phosphatase and do 30' at 56°. works better.

I've had the opposite experience from Fred -- I think it is quite easy to damage your vector while dephosporylating it, at least with calf intestine alkaline phosphatase (CIAP).

I don't have any published evidence for this, just that we seem to get much better cloning results by preparing the vector using a small amount of CIAP (0.5U) for a short time (30 minutes at 37°C) versus more for longer.

Of course, the absolutely critical step in all vector preparation, dephosphorylated or not, is to gel-purify the prepared vector before using it in the ligation (and gel purify your insert DNA too)...