Hi,
I have been trying for a long time several primers for qPCR amplification of EGFP. While EGFP amplifcation works well, so far all the pairs have been giving me a primer dimer, leading to some Ct value even in no template control. Does anyone have any suggestion on the primer pair that worked well for them?
Standardize correct annealing temperatures; according to your expected band size you may increase annealing time; while you are designing primers check the primers properties and design such primers that offers no dimer; there are different softwires available for the assessment of primer properties; also optimize the primer concentration; .
Wishing you have the problem solved.
There are plenty of factors resulting in no bands with just primer-dimer in your PCR!
1) You need to calculate the annealing tem. correctly. To this end practically, you can perform a touch-down PCR and make sure that your primers are correctly designed too.
2) Secondly, if your template is GC rich, you need to designate a bit of a long time for 'extension' steps and also use DMSO in your master mix. If you use DMSO, you can increase our tem. as well.
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