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Annealing temperature - (Mar/10/2010 )

Hi! Can anyone help me on what melting temperature, Tm to use for my ISSR primers? I am confused as to what melting temperature to use to determine the annealing temperatures (Ta) for my set of primers. In one article which used the same primers the Ta used range from 52-53.5 degress (depending on the primers) but doesn`t have an explanation how they were determined. Another article which used almost the same primers but for different species, the Ta was fixed to 52 degrees. The primers I bought came with specific Tm, which are way too low if I compute Tm using the formula Tm= 2(A+T) + 4(G+C). And if I compute the Ta (Ta=Tm - 5) using the Tm indicated in the primer labels, what I get are Ta values ranging from 36-54 degrees where most of the primers falling below 50 degrees.

Which options would be better for me:
1) Use the 52-53.5 (primer dependent) Ta;
2) Follow the article with fixed Ta which is 52 degress; or
3) Determine the optimum Ta for each primers by conducting a PCR run using a Gradient PCR machine (which will require much of my time and use considerable amount of reagents)?

Or if you have better suggestions they are most welcome.

Thanks!

-vhinoleigh-

annealingtemp has to be, 2 to 3 degrees lower then the Tm of the primers


vhinoleigh on Mar 11 2010, 12:58 AM said:

Hi! Can anyone help me on what melting temperature, Tm to use for my ISSR primers? I am confused as to what melting temperature to use to determine the annealing temperatures (Ta) for my set of primers. In one article which used the same primers the Ta used range from 52-53.5 degress (depending on the primers) but doesn`t have an explanation how they were determined. Another article which used almost the same primers but for different species, the Ta was fixed to 52 degrees. The primers I bought came with specific Tm, which are way too low if I compute Tm using the formula Tm= 2(A+T) + 4(G+C). And if I compute the Ta (Ta=Tm - 5) using the Tm indicated in the primer labels, what I get are Ta values ranging from 36-54 degrees where most of the primers falling below 50 degrees.

Which options would be better for me:
1) Use the 52-53.5 (primer dependent) Ta;
2) Follow the article with fixed Ta which is 52 degress; or
3) Determine the optimum Ta for each primers by conducting a PCR run using a Gradient PCR machine (which will require much of my time and use considerable amount of reagents)?

Or if you have better suggestions they are most welcome.

Thanks!

-susanna-

vhinoleigh on Mar 11 2010, 07:58 AM said:

Hi! Can anyone help me on what melting temperature, Tm to use for my ISSR primers? I am confused as to what melting temperature to use to determine the annealing temperatures (Ta) for my set of primers. In one article which used the same primers the Ta used range from 52-53.5 degress (depending on the primers) but doesn`t have an explanation how they were determined. Another article which used almost the same primers but for different species, the Ta was fixed to 52 degrees. The primers I bought came with specific Tm, which are way too low if I compute Tm using the formula Tm= 2(A+T) + 4(G+C). And if I compute the Ta (Ta=Tm - 5) using the Tm indicated in the primer labels, what I get are Ta values ranging from 36-54 degrees where most of the primers falling below 50 degrees.

Which options would be better for me:
1) Use the 52-53.5 (primer dependent) Ta;
2) Follow the article with fixed Ta which is 52 degress; or
3) Determine the optimum Ta for each primers by conducting a PCR run using a Gradient PCR machine (which will require much of my time and use considerable amount of reagents)?

Or if you have better suggestions they are most welcome.

Thanks!



Hi,

I think personally you are better off performing a gradient PCR. It will only take an hour to 2 hours and as they say "a stitch in time saves nine". its better to determine the best annealing temperature for your primers now rather than having to go back and repeat PCR and changing your annealing temp everytime. (which will end up wasting more resources and your time).

-wizzkid-

depends of course of how many sample you have...
Not every qPCR equipment can do the gradient PCR

wizzkid on Mar 11 2010, 07:21 AM said:

vhinoleigh on Mar 11 2010, 07:58 AM said:

Hi! Can anyone help me on what melting temperature, Tm to use for my ISSR primers? I am confused as to what melting temperature to use to determine the annealing temperatures (Ta) for my set of primers. In one article which used the same primers the Ta used range from 52-53.5 degress (depending on the primers) but doesn`t have an explanation how they were determined. Another article which used almost the same primers but for different species, the Ta was fixed to 52 degrees. The primers I bought came with specific Tm, which are way too low if I compute Tm using the formula Tm= 2(A+T) + 4(G+C). And if I compute the Ta (Ta=Tm - 5) using the Tm indicated in the primer labels, what I get are Ta values ranging from 36-54 degrees where most of the primers falling below 50 degrees.

Which options would be better for me:
1) Use the 52-53.5 (primer dependent) Ta;
2) Follow the article with fixed Ta which is 52 degress; or
3) Determine the optimum Ta for each primers by conducting a PCR run using a Gradient PCR machine (which will require much of my time and use considerable amount of reagents)?

Or if you have better suggestions they are most welcome.

Thanks!



Hi,

I think personally you are better off performing a gradient PCR. It will only take an hour to 2 hours and as they say "a stitch in time saves nine". its better to determine the best annealing temperature for your primers now rather than having to go back and repeat PCR and changing your annealing temp everytime. (which will end up wasting more resources and your time).

-susanna-