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NEB TM CALCULATOR PRIMER CONCENTRATION


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#1 cat42

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Posted 05 November 2014 - 04:08 AM

Hey all, I just have a question about the NEB Tm calculator as I plan on using their Q5 high fidelity polymerase.

 

Here is the link to the calculator: http://tmcalculator.neb.com/#!/

 

Where it asks for primer concentration I assume that, with my 10uM/L working primer concentration, the nM value will be 10000nM? Is this correct or have I used the wrong primer concentration?

 

Any help regarding this would be greatly appreciated. 

 

 



#2 dpo

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Posted 05 November 2014 - 06:11 AM

You have to put in the end concentration of your primer in the PCR mix, not the concentration of your stock solution. Normally, primer concentrations in PCRs are in the range of 100-900 nM (I mostly use 300 nM for standard PCRs).



#3 phage434

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Posted 05 November 2014 - 07:01 AM

These calculators are not very accurate, and you need to verify the reactions in any case. I'd suggest ignoring them and getting on with some experiments to determine optimal conditons. Start with a 55 annealing temperature. If you have a gradient cycler, this is faster and more accurate than worrying about calculators.






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