As you see in the picture attached that there are 3 graphs that have a short peak after 90 degrees and 3 others that have rising trend.
Do you know what I can do to optimize Real-Time PCR for my primers and what can be the reason for the results I gained?
You should have a single peak for a single amplicon.
Presence of multiple peaks are suggestive of primer dimer and non-specific amplicons.
It's possible the 1st peak is a primer dimer, the second peak the desired amplicon. The third peak has a very high Tm which may indicate that it is a long amplicon and may be an off-target amplification. You can try to do an agarose gel to see its size.
Should use primer design rules and software to minimize primer dimer and non-specific amplification. For example of some basic rules:
https://eu.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2013/10/21/designing-pcr-primers-and-probes
And use a primer design software with the parameters mentioned above, i.e.
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
No problem, remember primer design can be an art sometimes and even experienced users sometimes gets the dreaded multiple peaks. Have a read of this as well
https://eu.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2014/01/20/interpreting-melt-curves-an-indicator-not-a-diagnosis
- Badges
- Science topic