I am trying to phosphorylate both of my forward and reverse primers prior to the PCR. Primers that are 5' phosphorylated (theoretically) do not affect PCR since primer extension occurs from the 3' end of the primer. However, my several reactions with PNK'ed primers have been failing and when I used the non-phosphorylated primers, PCR works with no problem. I am assuming some chemical changes happening to the primer binding sites, preventing the primer from binding the template DNA. Anyone had this problem before? Any recommendations for troubleshooting?
Here is my recipe for PNK phosphorylation separate for each primer:
The mixture of 10uM Forward primer with 5’ end, 10X T4 DNA ligase buffer, T4 PNK (10 units) and Nuclease-free water 1) incubated at 37°C for 1 hour, and 2) incubated at 65°C for 20 minutes to inactivate the PNK enzyme.
You mention you are phosphorylating primers. Is it correct to assume these are ssDNA? If yes, check the sequence of the primers using something like Oligoanalyzer 3.0 (very useful, free on-line software for primer sequence analysis) for homodimer formation and to see if the 5'-end folds back on itself. What I am getting at is your 5' end could be tied up in a stable dsDNA region if the primer can form a stable homodimer or if the primer can form a stable dsDNA region as the primer folds back on itself to form a loop. If the 5'-OH is not 'available' to the enzyme due to either of these issues your phosphorylation will not work well or work very poorly.
Check to be absolutely certain the buffer you are using has ATP, and at what concentration.
If you determine that a stable homodimer and/or loop forms, you could try heating the DNA to a temperature above which the self-annealing will melt, snap-cool on ice, and then try the phosphorylation.
are you using dna ligase buffer as in your recipe or kinase buffer please and what is your source of phosphate for the reaction
Hi Paul Rutland , I am using T4 DNA ligase buffer as a substitute to the T4 PNK reaction buffer. The reason is that the T4 PNK buffer does not include ATP, which is necessary for the phosphorylation reaction (and T4 DNA ligase buffer has it). T4 PNK by NEB is my source of phosphate.
Thank you Bilgenur Baloglu I was concerned that without a source of phosphate the enzyme might scavenge phosphate from the oligo but that is not the case. I can not account for the inactivation of the oligos unless the buffer has become contaminated and is degrading dna, Possibly incubating some dna ladder in the reaction buffer and running it on a gel might test whether the procedure is degrading dna. Hopefully somebody can offer a better solution.
Check what paul suggested .
Try:
If it is blunt-ended then heat the buffer mixture itself for 10 minutes at 65°C and incubate it in ice chilled water then add ATP + enzyme followed by 37C incubation, this will help for priming
Hi Paul Rutland , I like your thinking process. The buffer is newly bought, and I always use filter tips in a so I would not think it is contaminated. I can still try the DNA ladder solution to make sure. Thanks!
You mention you are phosphorylating primers. Is it correct to assume these are ssDNA? If yes, check the sequence of the primers using something like Oligoanalyzer 3.0 (very useful, free on-line software for primer sequence analysis) for homodimer formation and to see if the 5'-end folds back on itself. What I am getting at is your 5' end could be tied up in a stable dsDNA region if the primer can form a stable homodimer or if the primer can form a stable dsDNA region as the primer folds back on itself to form a loop. If the 5'-OH is not 'available' to the enzyme due to either of these issues your phosphorylation will not work well or work very poorly.
Check to be absolutely certain the buffer you are using has ATP, and at what concentration.
If you determine that a stable homodimer and/or loop forms, you could try heating the DNA to a temperature above which the self-annealing will melt, snap-cool on ice, and then try the phosphorylation.
I am also using T4 DNA ligase buffer as a substitute to the T4 PNK reaction buffer. The reason is that the T4 PNK buffer does not include ATP, which is necessary for the phosphorylation reaction.
ATP degrades overtime. Old ligase buffer will not be efficient at phosphorylating your ends. Unless the buffer is alliquoted, i always add fresh atp to my buffers.
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