In general, people spend a lot of wasted time "cherry-picking" primer annealing temperature conditions when they get new primers. In my experience, the predicted Tm (that the company calculates for you) and therefore Ta is often quite different to the experimental one. Of course, you should try to design primers with T(m) within at least 5C - the close the better really - however, you should ignore the predicted T(m) altogether, and do a gradient PCR, across 50-70 for example, to determine the optimal annealing temperature of your primers. That is a big range, but will quickly give you an idea about how the pair of primers will behave. From there, you can optimise using a smaller temperature range if required - the result being (more often than not), an experimentally optimised annealing temperatures the will work great.

The T(m)-5C "rule" really should be considered an old concept, and shouldn't be used if you have access to a gradient PCR machine.

Ask yourself the question what would happen to the 63 tm primer if you anneal at 70? Would it be too hot for it to hold on? You have to go with the lower one. I agree with Stephen above, that too much time is wasted worrying about this problem. Designing the primers in the beginning is better than sorting out the Tm problem later.

If you are confused about the annealing temperature to be used, simply use a touch down PCR program to see which temperature is the most suitable to use both the primers. the Tm of the Forward primer is 70, so the theoretical Ta should be (Tm-5) or 65 in this case. For the reverse the Ta should be (63-5) or 58. If you have an Eppendorf or Biorad system please follow the instructions manual and set up the touch down PCR program. Start from an annealing temperature of 56 and then after every 3 to 5 cycles, increase it to a degree until it reaches 66.

you also can set up a gradient PCR where, each of the row in a PCR machine will have different temperature and you can set that up easily by following instruction manual. and then set your reaction mixture in several PCR tubes and put them in one hole of each row and use different temperature to amplify your products. depending on the band intensity you can easily infer which temperature is the best.

you can deduce the exact temperature required if you use any of the above mentioned protocols.

You may be able to go a little higher though, to prevent the other primer from annealing elsewhere.

Thanks you all

The annealing temperature must be 1-5ºC lower than the lower Tm of the primers. That is 58-62ºC.

Good luck!

In general, people spend a lot of wasted time "cherry-picking" primer annealing temperature conditions when they get new primers. In my experience, the predicted Tm (that the company calculates for you) and therefore Ta is often quite different to the experimental one. Of course, you should try to design primers with T(m) within at least 5C - the close the better really - however, you should ignore the predicted T(m) altogether, and do a gradient PCR, across 50-70 for example, to determine the optimal annealing temperature of your primers. That is a big range, but will quickly give you an idea about how the pair of primers will behave. From there, you can optimise using a smaller temperature range if required - the result being (more often than not), an experimentally optimised annealing temperatures the will work great.

The T(m)-5C "rule" really should be considered an old concept, and shouldn't be used if you have access to a gradient PCR machine.

You can play around 60

The annealing is Tm plus minus 5 degree. Use gradient PCR start from 66.5 degree this is the mid point of the tm of both primers. Use gradient of 5 degree it will cover the range of both primer and help you to find out the best annealing temperature for your desired primers,

@Artur - thanks for the correction - thats what I meant. Correcting the original post now

@Doyle. I have a query if Tm range =58-68ºC. Is any range for annealing temp (Ta). if we design primers we set Tm with the PCR product size,then is it possible to set annealing temp during designing of primers.

Hi I agree with Stephen and we usually test new primer pairs by gradient PCR. One reason is that not only the calculated Ta depends on the algorithm used but also the correct Ta heavily depends on the used PCR mixture.

Nevertheless I would always try to design primers with a similar Tm. If this is not possible there are some alternative oligo structures which can increase the Tm of primers (LNA - locked nucleic acids). Furthermore the amount of annealed primers is dependent on the concentration itself (described in papers about LATE-PCR). So it may be an alternative to use a higher amount of the primer with the lower Tm.

Good luck

@Neha, I am not sure if I understand you correctly… if the predicted is within that range that you have suggested, then in theory there will be a corresponding annealing temp range (is this what you mean).

@Norman is completely correct - most importantly, the PCR master mix influences the true annealing temp. Best bet is to do a gradient.

You can safely apply Ta even 72.

Important which method you have calculated the melting temperature of the primers. Moreover, taken into account when calculating the Tm of primers concentration than it is higher, the higher the Tm.

Finally, PCR product length, that the higher the length of the PCR product should be used the higher Ta applying, not paying attention to these Tm oligos.

http://primerdigital.com/fastpcr/m8.html

It mainly depends on the melting temperature of the primers. The annealing temperature is always slightly more than the melting temperature of the primers. It is because the primer has to melt before it start the annealing process for the continuation of the polymerase chain reaction (PCR)

I usually test new primers by gradient PCR, but about your case I think to be 60-63 oC (based on my experience).

I said based on my own experience !. because I had the same experience, Forward primer Tm: 71 and reverse primer Tm: 63, but both of them work well at 62.

it is depend on Tm of your primer and if you have not any refrence it is better that you use temprature gradient near primer Tm

Thanks you all

@Mohammad - how did your PCR go? Did you solve your problem?

Actually, I did gradient PCR 60-+ 10 and still have no amplification! Finally, I decided to change the primers.

@ Muhammad Faraz Bhatti

Is that annealing temperature is greater than melting temperature of primer? There is an information that, Ta is about 5 degree C below the Tm of your primer!

Reyhaneh Ravanbakhsh Can you tell me some annealing temp for this primer ?