I am working on honey bees, trying to isolate common bee viruses. I have specific primers, my samples are bee brood, varroa mites and adult honey bees. I am getting multiple bands even after doing gradient PCR and MgCl2 optimization. Please help!
It looks like primer-dimer for all of the reactions as well as non-specific amplification. What are the Tms for your primers? You might consider increasing the annealing temperature to help improve your specificity, or consider hot start PCR to make sure no amplification is happening before your cycles begin.
Another idea might be to use a touchdown PCR protocol - you start your cycles with a high annealing temperature to improve specific amplification, and gradually lower the annealing temp in subsequent cycles. This (ideally) produces a lot of specific product in the early cycles, which are rapidly copied in the later ones. This Roche protocol describes a combined hot start and touchdown method to increase specificity: http://www.roche-applied-science.com/shop/products/combined-hot-start-and-touchdown-pcr-protocols-optimize-amplification-of-difficult-targets
PCR additives like DMSO, betaine, and BSA may be helpful as well. Some concentrations that usually help for these are final concentrations of 5% for DMSO, 0.5 ug/ul for BSA, and 1M for betaine, but you may want to test a range of concentrations to optimize for your primers.
First of all you have to standardize the annealing temperature using gradient pcr adn use of pcr additives 5% DMSO or Glycerol can help to get clear bans for specific pcr.
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