Hi researchers
I am doing real time for some autophagy genes .
At first some of them didn‘t have any bonds or had primer dimer!! So I have reduced primer concentration ,increased the template concentration,changed annealing temperature and so on…
Now ,however most of them are corrected but some of them have primer dimers yet, even more than the real bond !!!
what should I do now?!Does any one have an idea?!
I am really confused
It is possible that you have primer-dimer contamination in one of your reagents so it is amplifying very well at the pcr stage. If this is the case change all of your reagents for new ones (including buying new primer stocks).
If the P-D is de novo and not contamination from a previous amplification then try using a hot start pcr enzyme for the pcr step if you are not already doing so
I suggest you make a primer matrix testing few combinations of concentration to test the best primer concentration, because sometimes just one primer concentration unadjusted can be the problem. You need too pay attention if your primers have quite degenerated, cause this can cause in dimer formation in high primer concentration.
The reason may be the expiration date of the primers or due to pollution during work.
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