Hi everyone
I have problems during my PCR reaction.
I got a specific band successfully around 590 bp using my primers. However, I got contamination on my primer. Then, I re-ordered the new primer with the same sequence but a different company. Unfortunately, I can not get the specific band.
The Tm primer from old and new are different.
Old primers, forward and reverse, have 69,7 and 71. 4 Tm. respectively
New primers have 59.7 and 60.1 (in the datasheet written that the Tm shown taken of Mg2+ and dNTP concentration)
When I ran PCR by the same condition as previous PCR (Ta 60 degree celsius), there is no band at all in my samples using new primer. Then, I ran PCR using Ta 5 degrees below the previous Ta, there are non-specific bands with a high molecular weight (higher than DNA marker).
I have no clue about this. I need your suggestion.
Thank you
I am very surprised that the Tm s are so very different. Check that the sequences ordered are exactly the same second time round. Also are all of the bases in the primers annealing to the template dna or are there added bases at the 5' end. Working at 10c below Tm is unusual. Check that your primers are not annealing over a polymorphism at, or near, their 3' end. If all these checks are good then try a temperature gradient to clean up your amplification or if you do not have a gradient pcr machine then run a sample with DMSO added at 1%, 2%....etc up to 8% dmso in the pcr mix to get rid of false priming and clean up your pcr
I agree with Paul, if you have checked the 2nd order contained the correct sequences you can run a gradient PCR to find the optimal annealing temperature. Are these high molecular weight bands well defined or are they more of a smear on the gel?
The DNA itself has the TM, however, you would need to calculate the TM according to the polymerase. the TM is significantly different between different polymerases. I think the manufacture website has a tool to calculate, something like this for NEB polymerase: https://tmcalculator.neb.com/#!/main
for another question: why no band by new primer?
you could try to use your old primer as a template to run the PCR by the new primers, Is it possible that the old primer somehow contains the positive template?
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