I m getting two bands in PCR product! I adjusted the temperature and time in cycles. The first band is the actual band which is around 518bp the second is not the one. Thankyou
At least the bands are well separated so you could purify from the gel if needed.
To clean up the pcr you could run an annealing temperature gradient (best option) or just keep increasing the annealing temperature and hope that the other band disappears. Use less primer...about half of what you are using as you have a small primer dimer.
You could also try a DMSO gradient 0f 1%, 2% up to 8% dmso in your pcr mix which will often clean up dirty pcrs
Try increasing the annealing temperature by 1 or 2 degree celsius than actual one
Due to non specific primer annealing, you should try to increasing the annealing temperature or use 5 to 10 pico mole primer to get a single band.
You can elute the upper band that one is specific band and can use for further process.
It means your primers binding at two sites. Your can purify your desired band and perform PCR again, and then perform gel electrophoresis if you need image containing only one band.
Moreover, you can run in silico PCR or check specific of your primers using NCBI primer BLAST option.
It would be better if you do sequencing to confirm the amplification of desired gene.
You're getting non specific binding from the primers; first try to rise the annealing temperature a bit and/or try using a lesser concentration of the primers.
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