So, here is what I am dealing with:
The Tm of the primers is 55 for the forward one and 56 for the reverse one. In -silico PCR produces 750 BP band, but in reality I get 3 bands. The third one looks like a primer-dimer.
Clearly the primers could be decreased and I used primers diluted to 10 uM each and I 1:3 dilution from it, which didn't give me any product. I tried 1:2 dilution today: this decreased band intensity and didn't get rid of the extra/lower band.
The question is where do I go from here?
-Gradually raise the annealing temperature?
-Will DMSO help?
-Increasing DNA concentration, while decreasing primer concentration?
Does Tm really matter? Should I just try different annealing temperatures?
Thanks in advance !
choose an annealing emperature that does amplify and run a pcr set with 0. 1%, 2% up to 8%dmso and at some dmso concentration the pcr will probably be a clean band. If your gene has pseudogenes or is one of a family of similar genes then touchdown pcr or new primers might be needed
I think You can perform primer gradient PCR to optimize the concentration of primers. Also, You can use pico molar concentration of primers. or use different gradient pcr such as with annealing and denaturation etc.
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