Can I simply add a random sequence to the 5' end of my PCR primers and use this as sequence primers? The degenerate primers are designed against a highly conserved part of G-alpha subunits and will be used against Dinoflagellate cDNA.
A second question: The melting temperature of the forward PCR primer is 49 degrees and the melting temperature of the reverse primer is 61.3 degrees. The sequences are only 18 bp long. Can I add a random CG rich sequence to increase the melting temperature of my forward primer and run the first 5 cycles with a low annealing temperature, followed by a higher annealing temperature (around 55) for the rest of the cycles to make sure the melting temperatures of both primers are closer together?
Thanks in advance!
You can either use Automated Sequencer for your sequencing, if available or send your sample to sequencing lab, which is specialized for sequencing.
Our experience of this has been that it generally doesn't work very well trying to directly sangar sequence PCR products with degenerate primers as a template. We nearly always have to E.coli clone these and sequence them from the plasmid (M13 primers on PGEM-T-easy or Topo-TA plasmids usually). Its a pain but we've just never gotten it to work otherwise. With your second question I've never really tried to get TMs on primers to match - we tend to just see if its going to work across a range of temperatures and or re-design the primers. You can spend an awful lot of time fiddling with primers to get them work and nowadays it often proves easier and cheaper to just try a new set of primers (they really aren't that expensive any more)
Thanks for your answer Rachael. Do you maybe have a useful cloning protocol for me?
The success of sequencing primers with degenerate primers depends mainly in the obtained PCR product. If you obtain a single, clean and well concentrated PCR product, you can try sequence it, the time and money spent s very low. I did it several times with good success rates. If you obtain more than one band, even if you purified the product from the gel the sequence is usually of bad quality.
Might I ask, how did you solved your problem with sequencing of PCR product using degenerate primers? I have the same problem at this moment and a have no possibility to use E. coli clone.
@Slavka,
I send the PCR product for sequencing with all the possible primer options but that didn't work for me (it turned out that still one of the bases was different). So I cloned the PCR product into E. coli using TOPO TA cloning, it was very easy but you do need to buy a kit to do so. Good luck!
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