primer Tm - (Jan/26/2007 )

I have recently purchased primers from Invitrogen. The product sheet comes with two Tm's for each primer. Which do I use to calculate the annealing temp for a PCR reaction. The tm's are 78 and 56 for the forward primer, and 76 and 55 for the reverse. Please confirm which to use . The current PCR conditions use 67 degrees for the annealing, but the product only amplifies sometimes. The DNA is genomic of good quality.

Thanks
SYBR

Is it the salt adjusted Tm and the basic Tm or even base stacking Tm?

Not really sure which one but usually I will follow the basic one.

I am also curious about it. Cheers

Plug the sequence into a Tm calculator if you have the sequences, that will tell you

there is basic tm ie the tm if the primer was place in distilled water

there is salt adjusted tm, ie the tm of the primer in a salt solution of a certain molarity.

The tm you want to use is the salt adjusted tm, as the PCR reaction works in a salt buffer.

Now there are two methods for calculating tm..I can't remember their name. In anycase, they give similar though not identical answers. Both claim superiority over the other... I have no idea which is better. So when you go looking on the net, pick one and don't be too worried if another calculator gives a different answer. Just use the same calculator for both primers


I use Northwestern's calculator

And note, the annealing temperature of your primer is calculated only from the tm of the part of the primer which actually binds to the template. Your annealing temperature is not the tm of the entire primer - 5 Celsius

Thanks very much. In that case the Tm I should be concerned with is 56 and 55 degrees. What do you recommend I use for the PCR annealing temp? 5 degrees below? The current conditions are 67 degrees, but the product is only present sometimes.....Any reason why this happens.


Thanks,

SYBR

just a random question.

what if I add TE buffer into my primer? which one should I pick?

How about the base stacking Tm?

I would say the salt adjusted tm.

as long as your PCR buffer does not change, the tm would reflect the salt concentration of the said PCR buffer.. so in otherword the salt adjusted reading.

And yes, you can change conccentration of salt and thus alter the exact tm of the primers in the PCR mix.