I am currently attempting to conduct PCR analysis using GoTaq polymerase (GoTaq Green Master Mix, Promega) and small primer sets from IDT that are seven basepairs in length.
My problem is that these primer sets have very low annealing temperatures ranging from 0.0 deg C to 10.1 deg C, yet the optimal temperature of extension for the GoTaq polymerase ranges between 72-74 deg C.
While I have attempted a slow increase in temperature increments in order for my primers to anneal and potentially be extended, the high temperature required for efficient extension bypasses my melting temperature (tm) by over 60 deg C, thereby resulting in the unbinding of the primers.
Does anyone have any recommendations or advice on how to allow my primers to anneal and be extended before they unbind from the DNA due to the relatively high temperatures used for the GoTaq?
My PI recommended that I oversaturate my samples with primers. Would this be effective? A concern I have is that the dsDNA I am using in my samples may easily re-anneal at these low annealing temperatures following the unwinding step of ~94-96 deg C.
If anyone is curious, two primer set I am currently using is 5' - AAGCTTG - 3' and 5'-CAAGCTT-3
The biggest problem right from the off is that your primers are perfectly complementary. Considering in this PCR reaction mix the proportion of primer compared to complementary target templpate sequence will be extremely high (I imagine) the rate of cycle upon cycle amplification will be fairly low as the primers will tend to bind to themselves.
Personally I think it's virtually impossible to generate a standard PCR protocol using these primers and a thermostable taq polymerase for all the reasons you mentioned.
There might be a way using Klenow fragment and manual movement of sample to various temperature water/ice baths and addition of fragment every cycle.....if you want to go proper PCR pioneer retro!!
Random hexemers are frequently used to generate cDNA libraries and will anneal at room temp (20-25 deg C) so yours should too.
To try and generate ssDNA template heat the DNA to 95 deg C then cool rapidly in an ice bath (dry ice and solvent perhaps!). Whilst some will re-anneal this should leave you with enough ssDNA onto which you can prime.
Klenow Fragment will amplify at room temp but this is thermolabile so you would need to add fresh fragment at each cycle.
I'm guessing by the lack of answers this is not an easy problem to overcome, perhaps you might need to ammend your initial aims!!
Why are earth are you trying to use 7-bp primers that are complementary to each other? Go back to the drawing board and design new primers that are longer and don't pair with each other. What template are you trying to amplify?
Generating ssDNA templates would not help solve the annealing problem.
I'll suggest you use longer oligonucleotide primers that are not complementary.
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