I am attempting to amplify nirS and nirK in some salt marsh sediment samples for my Masters thesis studying denitrification. My nirS gene is amplifying great in both PCR and qPCR. In the literature, nirK seems to amplify well at an annealing temperature of 57-58 C. However, when performing PCR, the target sequence amplifies well in my bacterial strain control at 50 C annealing temperature using HotStart Polymerase. I switched over to qPCR using 2X SYBR Green polymerase and increased the annealing temperature to 55 C because that was the standardized protocol in the lab. It worked incredibly well for nirK except upon inspection of a gel I have a beautiful band at the wrong bp length (700bp and my target sequence is 160)! No other bands show up at the correct length. I could lower the annealing temperature down to 50 C but that is incredibly low considering that I believe no specific amplification is occurring already at 55 C. I could also increase to 58 C but amplification doesn't occur at that temperature either. What should I do?
Could it be that you are amplifying genomic dna to give the larger product and that you have little expression of the 160 product in your samples. Sequencing or restriction digest of the 700bp fragment should give you an idea where it originates from
The melting temperature of primers depends on the buffer that you are using for the PCR reaction. This website calculates melting temperature for primers for common PCR buffers: https://tmcalculator.neb.com/
If you are unsure about the melting temperature, then I would recommend looking up touchdown PCR (TD-PCR). This is a normal PCR except the melting temperature is lowered by one degree after each cycle, which allows you to get specific amplification of your product. If you still see a band of the incorrect size after doing TD-PCR, then either the primers are poorly designed or there is contamination of some other DNA that is being used as the template.
Ciaran Daly Thank you for providing this information. Interestingly, the annealing temperature is recommended at 58 C for the primer pair I am using in multiple papers who also use the same polymerase. When I use online calculators I get a crazy range of optimal annealing temps (ranging from 49 - 60 depending on the calculator I use). I will look into touchdown qPCR becuase other papers have tried this approach as well. If not then there is another primer pair I can use that is reported as successful in many publications.
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