I used two different thermal cyclers from two different companies for the same sample and the master mix, under agarose gel electrophoresis there was a totally different result.
Hi, what I mean is that in optimization, using one thermal cycler, at 63 C for example, there was one band (160bp which is size of my target ) and there was no non- specific band).
but the same conditions were used in other thermal cycler, at 63C, there was just one band with 500 bp (non-specific band), and I couldnt see any amplification for my target
Did you repeat your experiment? Did you use exterior places in the cycler? Cheers, Nadine
Yes it could. I have seen it first hand with Eppendorf Thermocycler while making NGS libraries. Instrument has 3 different settings and basically slows down the ramp / or clock counter so you have enough time to reach to the desired annealing temperature. The standard settings are way too fast and that is not good for the amplification of GC rich sequences. Hope that helps
Yes, If I have to optimize PCR I usually do three reactions - one with Forward primer only, one with Reverse only and one with both. I am trying to isolate which primer is causing the problem. If primers are OK in 1 and 2, then I focus on the annealing T. I have seen counter intuitively that sometimes too high annealing could cause non-specific amplification as well. God luck.
Offcourse it makes differents, as "Ramp Rates" vary from companies to companies and versions to versions. Once you optimize your protocol on one thermal cycler continue to use on the same.
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