I need to design cloning pcr primers. I have made forward primer of 65 nucleotides and reverse primer of 32 nucleotides. Though the sequences from gene of interest for both primers are of same number,along with that forward primer has gc clamp, re site, kozak sequence and flag tag . Reverse primer has gc clamp and re site along with sequence from gene of interest. Would they be able to do amplification?
You will have to use an annealing temperature appropriate to only the sequence specific bases at the 3' end of each primer for the first few pcr cycles and have some extra bases 5' to the RE site to enable RE digestion. Longer primers may suffer from secondary structure issues so using a high GC buffer with your enzyme for amplification
It's a standard enough protocol that there are no inherent reasons this won't work. As Paul Rutland said, you ignore the part of the forward primer that doesn't anneal to the template when considering the annealing temperature. Plenty of online tools to do that calculation for you. Just use the parts of the primers that are complementary to the target gene as the input.
Good luck!
I'm gonna advise you redesign your primers, because primers with nucleotide base pair above 30 most times is an issue. And higher GC content here is also going to be an issue during the pcr reaction.
I will also advise you carryout an Insilico pcr using the genome database of your species of interest for optimization of your pcr profile.
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