Loss of phosphorylation after gel purification? - (Feb/27/2007 )

I am currently having some problems with a PCR fragment I amplified with phosphorylated primers. PCR yield was good but an unspecific fragment was present (even after optimization of the program) so I decided to purify the TAE gel with my fragment.
I re-ran the purified fragment on a gel and a clean fragment was visible in fair amount.
When blunt cloning in a dephosphorylated vector I am unable to get the right clones. Therefore I wondered if it is a possibility that I somehow lost the phosphate group on my PCR fragment after gel purification. Does anyone know if this is possible? All suggestions welcome. tnx

It sounds like problems with ligation. Actually blunt-end ligation are more difficult than sticky ones and I've never heard about dephosphorylated PCR product after amplifying with phosphorylated primers (unless you use a phosphatase, of course). Have you tried with different conditions for ligation? Even manufacturers give recomended different conditions when it's a blunt or sticky end ligation. And what about the rates verctor:insert? Have you tried with different rates? What says controls about dephosphorylation? What is the colonies rate between ligation and dephosphorylation control? I'd suspect of all this possibilities before the dephosphorylated PCR product theory.

Good luck!

I am sure this was taken into consideration, but are you sure the Taq you have leaves blunt-ended PCR products? I have had trouble with ligating gel-isolated fragments, I did not diagnose the cause, but when I used low melt agarose and did in-gel ligations everything worked out... maybe worth a shot?

Thanks for the replies. I did indeed take into considerations the ratio's and controls etc.. but nothing different than usual (I do quite regularly blunt ended ligations). The polymerase is called Primestar (Takara) and generates goo amount of blunt ends and is an excellent enzym in general (I recommend it for all difficult PCR's).
I am now trying to optimize the PCR program so that I can skip the gel purification step and simply purify over a collumn. If this doesnt work i will definetly try the low-melt gel thanks for the tip. When I first started cloning I had also problems due to the gel caused by use of TBE buffer instead of TAE so better always to use TAE.