How can we distinguish between Primer dimer and non specific amplification using gel electrophoresis and melt curve?
Hi Wajd,
The primer dimer on the gel would be the one at the bottom of your gel. In your melt curve, the first peak early on around 60C will be your primer dimer. Usually the target / specific amplification should be around 80C.
To get rid of your primer dimer, you should repeat your experiment 3 to 4 more times in triplicate and each time increase your annealing temperature by 1C. This would also help against non-specific amplification.
Good luck,
Ali
Hi Wajd Amly....
If you want to distinguish between primer dimer on gel then
1. Amplicon size: the size of your amplicon is determining parameter where the amplicons on gel are specific / nonspecific or primer dimer. Other than expected size of amplicons are non specific. However if it is primer dimer it can be identified by max amplicon size of < 45 bp in length as, generally primers length will be 20 to 24 bp. If on the basis of band size, if you are not able to distinguish between primer dimer and expected amplicon size then increase the gel % or use the recommended gel percentage for the ladder which your using.
2. You can also distinguish among primer dimer / amplicon and nonspecific in real time pcr using melt curve. The melting temperature of primer dimer is generally low compared to amplicon or non specific. If you select single gene dissociation curve it will show only single peak corresponding to specific amplicon or else more than onle peak will be seen at different temperature in single gene. So this will give you clue about primer dimer nonspecific and specific amplicons.
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