# Difference in Primer Melting Temperatures - Maximum allowable Tm difference? (Feb/26/2009 )

Hello,

What is the maximum allowable melting temperature difference between forward and reverse primers?

I've obtained a pair of degenerate primers with a 4 degree difference in melting temperature (e.g. 55 vs 59). (Temperatures were calculated by the manufacturer).

Could this be a problem?

I'm planning on running a temperature gradient to determine the best annealing temperature; can anyone please suggest what annealing temperatures should I try?

Many thanks!

-rustyshackleford-

rustyshackleford on Feb 26 2009, 02:57 PM said:

Hello,

What is the maximum allowable melting temperature difference between forward and reverse primers?

I've obtained a pair of degenerate primers with a 4 degree difference in melting temperature (e.g. 55 vs 59). (Temperatures were calculated by the manufacturer).

Could this be a problem?

I'm planning on running a temperature gradient to determine the best annealing temperature; can anyone please suggest what annealing temperatures should I try?

Many thanks!

I do not know the maximum allowable difference. But the maximum difference that I would allow is 2 Celsius. In situations where there is a difference in primer tm, use the lower tm. I would try using a melting temperature of 52 C.

As for a temperature range
50C, 52C, 54C, 56C,

Could this be a problem, yes it can. Will it be a problem... well you will have to give it try and find out.

-perneseblue-

Hey

Hey

I have gone upto a difference of 4 degree C and generally use 4 degrees less than the lower Tm for annealing.

TC

perneseblue on Feb 27 2009, 06:40 AM said:

rustyshackleford on Feb 26 2009, 02:57 PM said:

Hello,

Many thanks!

I do not know the maximum allowable difference. But the maximum difference that I would allow is 2 Celsius. In situations where there is a difference in primer tm, use the lower tm. I would try using a melting temperature of 52 C.

As for a temperature range
50C, 52C, 54C, 56C,

Could this be a problem, yes it can. Will it be a problem... well you will have to give it try and find out.

-T C-

rustyshackleford on Feb 26 2009, 02:57 PM said:

Hello,

What is the maximum allowable melting temperature difference between forward and reverse primers?

I've obtained a pair of degenerate primers with a 4 degree difference in melting temperature (e.g. 55 vs 59). (Temperatures were calculated by the manufacturer).

Could this be a problem?

It should be ok. I had used a short forward primer(Tm 50) and a long reverse primer (Tm 65) because I want to add some extra sequence at the 3' end. The annealing temp was 55, and the amplification was good.

-WHR-

Hi all,