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Annealing temperature for PCR


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#1 Meng Li

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Posted 26 March 2013 - 01:00 AM

Hello and good day!! I am a beginner in PCR. I would love to know on the calculations of annealing temperature of the PCR for the primers I am using.

Forward: 5' GGA GGA TTT GGA AAT TGA TTA GTT CC 3'
Reverse: 5' CCC GGT AAA ATT AAA ATA TAA ACT TC 3'

I have been searching in Google for annealing temperature calculator and there were so many versions. It is kind of confusing as there is differences between the calculated annealing temperature. How should I know which one is the best to rely on??Is there any specific calculators can be used provided that I do not have the concentrations of the primers and salt I am using?

Thank you so much for the time and hope to hear from you all soon.

Cheers
meng

#2 GNANA

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Posted 26 March 2013 - 04:35 AM

i go with online tools to analyse the primers, like primer3 (NCBI primer blast), or Oligo analyzer (http://eu.idtdna.com.../OligoAnalyzer/), ...

obviously values of both cannot be same as the calibration for each tool differs, however, for ur primers F-58.5, R-53.5 as per NCBI primer blast, and F-54 and R-49 as per the oligo analyzer (these values are with the tool's default setting )...but i think that is fine, though the primer tm is bit low but not bad. i would first go with 53 and/or 55 as annealing for these primers, if i have a problem then i would do a gradient.

good luck....
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#3 Trof

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Posted 26 March 2013 - 06:34 AM

You can't relly on anything, it's usually arbitrary, depending on your mix composition, primer length and many other factors you may get different optimal Ta. What works for me is the Tm calculation from primer3 minus 4-5 degrees for the first attempt. Have to note, that I usually also design my primers in primer3, ideally for 65 degrees Tm and use them on Ta 60.

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#4 Rahaf_Issa

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Posted 26 March 2013 - 01:22 PM

What are your melting temperatures? You can use the following equation to determine annealing temperature: Ta = average melting temperature of both forward and reverse primers then subtract 3 degrees from the total.

However, its best to run a gradient PCR where you subtract 1 degrees from your primer that has the lowest Tm and then run a PCR covering a 10 degree range. For example, if the Tm of your forward primers is 60 degrees and reverse is 58 degrees, then you would do gradient PCR using temperature range of 47 to 57 degrees. That way you can determine the exact annealing temperature.

#5 Meng Li

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Posted 26 March 2013 - 06:20 PM

What are your melting temperatures? You can use the following equation to determine annealing temperature: Ta = average melting temperature of both forward and reverse primers then subtract 3 degrees from the total.

However, its best to run a gradient PCR where you subtract 1 degrees from your primer that has the lowest Tm and then run a PCR covering a 10 degree range. For example, if the Tm of your forward primers is 60 degrees and reverse is 58 degrees, then you would do gradient PCR using temperature range of 47 to 57 degrees. That way you can determine the exact annealing temperature.


Hi! I calculated my primers' Tm from this equation: Tm= 81.5+0.41(%GC)-(675/N) where N is the total nucleotides in the sequence.
From there, the forward primer's Tm is 71.3 degrees and reverse primer's Tm is 66.6 degrees.

#6 phage434

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Posted 26 March 2013 - 07:11 PM

You can calculate all day, and look up any number of formulas, but that won't make your PCR work. Try it. I'd suggest starting with a 55 anneal.

#7 badguy

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Posted 27 March 2013 - 11:28 PM

The formula I used is Ta= 2(A+T)+4(G+C) - 2
I was taught to use that formula and so far so good.

#8 Tabaluga

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Posted 28 March 2013 - 08:52 AM

phage434 is right. There is any number of more or less accurate. Just use one to determine a "starting point" and then do a gradient +/- 5 °C.

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#9 bigudukaz

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Posted 04 April 2013 - 01:51 AM

The formula I used is Ta= 2(A+T)+4(G+C) - 2
I was taught to use that formula and so far so good.


I personally start with -4 (instead of -2) and had no problems so far ;)

Best option is to run gradient - you will get best results and those results will be for your primers, master mix (or components of your choice) and everything else.

So you just have to try and see ;)




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