I got primer dimers for my PCR. Then I tried to use hot start master mix for PCR and ran the gel. I got multiple bands for hot start PCR product. What is the reason for this.
Probably the answer is two fold. You may be using too much primer which often leads to primer dimer. Try 1/4 the amount of primer and also run an annealing temperature gradient. Often primers used at too low a temperature will anneal in lots of places and give many bands. As the annealing temperature is raised the number of wrong bands drops and hopefully there will be a higher annealing temperature that gives a clean ,single product. If this fails then try a DMSO gradient with the same sample dna and into each of 8 tubes add1%, 2%, 3% etc up to 8%dmso and the pcr should get cleaner with increasing dmso concentration and possibly not work if the dmso concentration is too high
The occurrence of multiple bands in PCR might be due to several reasons. You can try to reduce the final primer concentration in the PCR reaction. Low annealing temperature can also cause multiple bands as you mentioned. Better to run a series of 10-15uL PCR reactions in a temperature gradient PCR. Then you can select the best annealing temperature for cloning PCR.
There are several reasons you can end up with multiple bands on PCR. Common causes are too many cycles, annealing temperature too low, annealing or extension time too long, too much primer in the reaction, incorrect ramping speed, or too much MgCl2. I would double check the calculation on the concentration of primers and MgCl2. Then I would try to increase my annealing temp. Not sure how many cycles you went but if you go much above 35 you increase the chances for multiple bands.
Paul Rutland Thank you for your support. Helpful.
Sarah Zilka I used 35 cycles and reduced annealing temperature and primer concentration. Still got primer dimers.
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