#PCR is a very common technique many of you would apply, yo know whats even commoner? non-specific bands in your PCR product!
What went wrong?
a. Wrong primer design
b. Low annealing temperature
c. Contamination in the template DNA, could be other DNA or chemical contaminants
d. High #GCcontent
Solutions?
1. Do a gradient of #PCRadditives keeping all other reagents and conditions unchanged (3% #DMSO or 2.5M #Betaine usually works good with high GC content templates).
2. Increase the annealing temperature upto 70C (Temperature-gradient), but this might bring down the intensity of the desired product.
3. #TouchdownPCR protocol maybe followed wherever possible depending on the instrument. Here the initial annealing temperature is kept more than Tm and brought down gradually in subsequent cycles of the reaction.
4.1 μg/mL of #grapheneoxide has been shown to effectively enhance the specificity of multi-round PCRs.
5. #Primer redesign may be the next step if the issue persists.
6. Lastly you might elute the non-specific band from gel and purify and sequence it to confirm its origin, and approach with primer redesigning.
Hope it helps!
More to munch on:
Comments