Phosphorylated primers - (Feb/12/2010 )

I have ordered phosphorylated primers two months back from invitrogen to perform site-directed mutagenesis. I have performed all the necessary purification steps, ligations, and transformations. I don't see any colonies of my LB amp plates. I ran my ligated product on the gel and haven't noticed a supercoil band. so, I think that the possible reason for not seeing any colonies is because my vector has not self-circularized. I beleive that this could be because of the degradation the phosphate groups on the primer ends. The transformations of positive control and efficiency of competent cells are great (see lot of colonies). I have tested my T4 DNA ligase by ligating 1 kb ladder and that's working well. Do you all think that phosphate groups have degraded as well? If they have degraded, what does it mean by degradation: no more phosphate ends or the intensity (I can't seem to think of the right word) has gone down? I know that I can't overphosphorylate something, so if it is degraded (but phosphate groups are still present), do you think phosphorylation with PNK work at all?

Please help me out,

Thanks,
Ougadougou

So, are you doing a PCR with phosphorylated primers followed by blunt ligation? What enzyme are you using for th PCR?

phage434 on Feb 12 2010, 05:08 PM said:

yes and I'm using Pfx polymerase from invitrogen

How are you preparing your vector? Can you do a control blunt ligation of the vector only and get transformants?

phage434 on Feb 16 2010, 02:49 PM said:

The vector was already made and was given to me. It has got the backbone of pBSK vector. All I am doing is creating a mutated site via mutant primers. The primers abut each other with one going in the sense direction and the other going in the antisense direction. Once the primers have been completely amplified, the ends of the primers which contain phosphate groups are ligated using the blunt end ligation protocol. My positive control seems to give me colonies after I transform it.

I have another question: If I digest my control plasmid and ligate it again, would I be able to see a supercoil band if completely ligated, or is supercoil generally not produced upon ligating a digested product.

This is just a guess, but you may have dramatically too much of your annealed oligos in the ligation reaction. The oligos will self ligate, and ligate to the vector, and it will be very rare for a single oligo fragment to insert and recircularize. Calculate the amount of oligo DNA you need to create an equimolar ligation reaction (1 molecule vector, 1 annealed oligo pair) at relatively low concentrations (say 20 ng of vector in a 10 ul reaction). Your main problem (eventually) will be too many religated vectors with no insert, and no good efficient selection for the one you want. Think about how you will select for correct colonies. A phosphatase treatment of your vector may help reduce religation, but it can be tricky to optimize. Oh, and you should be using quick ligase buffer, or another buffer with PEG to enhance blunt ligation.