Hello everyone!
I got a similar query!
I have calculated Tm for my primers and those were in the range of 58-62. However, when I received from the company, those were in 53-58 range. And now the annealing was not happening at 49-50 range. I was performing gradient PCR and the annealing was happening in 41-43. I need a better suggestion to optimize the annealing temperature, as I'm interested in performing q-RT PCR and such lower temperature annealing is a problem for my comparison with HKgenes.
Thank you for your input.
Looking forward to your valuable inputs.
Hi there
there are many algorithms for computing this factor: none are completely accurate and all tend to give results which differ by 2-3C
Based on what you have said, setup 10 reactions between 60C annealing and 50C annealing, each differing by 1C (hence why 10)
Hi Rajarajan,
Sometimes the TM and annealing temperature that companies say are wrong. Actually not wrong, but not working in practice, that is theoritical. Last year I faced with the same problem, and I tried 2 different gradient PCR. You can try anothet gradient pcr with the annealing temperature between 50-58 and 55-63. If you see any bands in any of the pcr, you can do another pcr to verify and sure about the temperature. For example if you see a band in 50-58 pcr in 52 C, but the band is not that bright as you want, then you will try 51-54 range.
I hope and sure this will work. If not, you can ask again. Good luck for your work.
Hi there
there are many algorithms for computing this factor: none are completely accurate and all tend to give results which differ by 2-3C
Based on what you have said, setup 10 reactions between 60C annealing and 50C annealing, each differing by 1C (hence why 10)
Nurefşan Cirik Laurence Stuart Dawkins-Hall Thank you very much for your timely recommendation. I will try the gradient PCR in higher range as recommended.
Hi Rajarajan,
By my experience, you would likely get the PCR products somewhere from 58-65. You can try adding small amounts of MgCl2 or DMSO or glycerol. MgCl2 would be the better option. And I hope to that you have the template in proper amount. If you are using a mastermix, you can try varying the template concentrations and additional MgCl2. Whereas if you are making the mastermix yourself, try varying the concentrations of different components.
Hope this helps.
Hi Arun
I like your answer but keep in mind that Glycerol, DMSO and MgCl(2) do completely different things
1. MgCl(2) increases the activity of the Tax: Thus it can boost amplification efficiency but has no effect on annealing temp. Too much can lead to extra bands
2. Glycerol increases enzyme stability and thus is useful with 35+ PCR cycles and/or denaturing temp > 96C (if GC content of target > 60%)
3. DMSO melts secondary structure in Gc rich DNA
I would say that an annealing temp gradient plus 5% DMSO might be useful
If you find very weak bands at all temp add a further 5% Glycerol to to PCR reaction; > 35 cycles and denaturing temp of > 96C
If that fails, try adding 0.5mM MgCl(2) to your PCR reactions
DONT use all 3 all at once
I think that you have a very large difference between theoretical and practical annealing. If your problem is not already solved check very carefully that the primer sequences as made align exactly with the template sequence and also check that there are no known snps lying under the primers .A mismatch base will lower the annealing temperature quite a lot. It might even be necessary to design a new primer pair outside your current primers
@Laurence Stuart Dawkins-Hall
Thanks for the beautiful supplement.
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