I ran a gradient PCR and got multiple bands on all the temperature regimes that I had programmed. Why was this the case? How can I correct this so that I get the optimum temperature?
Charles,
Have you run a melting curve after your PCR? Sometimes the melting curve can cause extra bands that are not present without the melt.
Also you can try to do a primer titration series for your primer pair if you are stuck with your primers and have checked that they are super specific.
If you get multiple bands, you have non-specific amplification. This is probably due to a primer problem. You need to have primers that ard Td matched (there are a number of online and offline softwares that can calculate the Td of you 2 primers ... and check that will not form primer-dimers). As Sailesh sais, touchdown can help here, but if it does not work, I'm afraid thatyou will have to redesign your primers.
Thank you all for your advice. I will try to do what you have suggested
sequence specifics can also result in generating the multiple bands, secondary structures, and amount of the template DNA that you use. As little as 10-15 ng of template in 10 ul reaction can be seen on the gel. Check for hairpin in your primers, analyze your sequence if GC or AT rich and stable hairpin or stem-loop structures. Than increase the Tm to melt those or use less template , or play with elongation temperature. For AT rich regions- reduce the elongation temperature and for GC add additives (CES mix- DMSO, Betain) or increas teh temperature.
If you need the amplified DNA for cloning and you see your desired band on the gel just gel-ectract the fragment and use further.... and verify by sequencing
Some time optimization may take a lot of time and efforts as well as materials ....even to experienced users...
THink about your final goal......
If you need primers for RT-PCR - than that is critical to eliminate the primer dimers and muliple amplicons.... to avoid that look at Roch primer library (already designed and tested primer sets for almost all genes and good for RT PCR)
Good luck!
i think you shoud dilute template dna. with a ratio 1 to 5. or 1 to 10. with distilled water
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