Dear Arwa, both Tm and Ta of primers are very often misunderstood. Apart from the above discussion and knowledge input for your query I would like to impart here some more basic info, which one should know when dealing with the primers.

You, till this point already have the idea regarding primer Tm and Ta as per their normal  definitions. 

Now, what you must know that a primer's Ta should always kept less than its Tm. So lets assume in reverse that for a primer you have Tm less than the Ta, so every time when this temperature will reach your primers will always be in melted stage and there will be no annealing to the template at all, as the Ta is still higher than the Tm. So its very simple and logical without complexities. There will be no amplification or if anyhow there is the product, the yield would be negligible. For example if Tm is 60 C and Ta is 65 C so how come your primer will anneal to the template. I hope you got it.

A perfect ct curve will only appear if your designed primers are specific in terms of their sequences, Ta and the Tm. One of the major cause of double peaks is also poorly designed primers, though there are several other factors too are involved for multiple peaks.

In general as per primer guidelines, a primer Ta must be 3 - 5 C below its Tm is excellent no doubt.

Ta and Tm both are directly proportional to the primer length. Ta above 72 C not recommended at all and also a primer pair with a very low Ta may anneal non-specifically giving false products not the actual ones.

And important to know for the primer Tm, that both the F and R primers if are not having the same Tm (which is also fine), so their Tm difference should not cross above 5 C, as this will cause unequal extensions and often a failed amplification. Example, if F primer Tm is 55 C so R primer Tm by no means should cross 60 C. It must lie within the 5 C range. Ideal primer Tm by guidelines is 52 C - 63 C.

Highly rich  GC % also not good for the primers, as such primers cannot denature easily even upon reaching their Tm, so you will definitely face issues in your melt curve appearance, which are incomprehensible and nobody wants that. So follow the ideal GC ratio range, which is usually from 40 - 60% GC.

I do hope your concepts are more clear now. 

Sincerely.....

The best Tm depends on many factors (Mg, dNTP, template and primer concentrations, heat transfer kinetics between the block and the solution, renaturation kinetics of the template, etc) so it is impossible to predict it with 100% accuracy. Use the predictions of primer design programs as a starting guide and tweak your PCR parameters along the way as necessary.

Thank you, Alejandro Martin, for your answer I appreciate your help, but I want to ask you more if you excuse me, the Tm of primers doesn't give any indicate about the annealing temp. whether its low or high isn't important the important is whether gave perfect curve of Ct or not, right?  

Arwa, in many cases, primer designing programs are already pre-loaded with parameters that allow one to generate primers that have a Tm of 60C for qPCR applications.  But as Alejandro has mentioned, Tm is a function of several other factors: mastermix ionic strength, Na+ and Mg++ concentrations, and the primer concentrations themselves.   Tm is defined as the temperature at which 50% of the primer is annealed to target.  For a Tm of 67C, (if the algorithm you used already took some basic general parameters into account during design), would indicate to you that 67C should already be at or near the suggested annealing temperature for qPCR application. Sounds like you have high G-C content in your primer(s)?

Generally, you'd want to design primers with Tm values nearer 60C in most situations.  Depending on the program and/or algorithm you used to calculate your Tm values affects the calculation as well.  And, yes, if you get great results; amplification curves and acceptable Cq values etc., then you have indeed achieved your ends - especially if you have no primer dimers or non-specific side-reactions.

A possible benefit of higher Tm values is that primer dimers and non-specific reactions would be discouraged, and taq polymerase would be more active (since its optimal temp of operation is ~72C - given its genesis from hot spring thermus aquaticus bacterium in the first place)...

Thank you, Jack M Gallup, I appreciate your valuable information, yes our primers with high GC content and it'll try to increase the temp. since it gave me two peaks which indicate the reaction didn't go well, thank you again.

Dear Arwa, we use ABI web tool to calculate Tm values of the primers for Real-Time PCR and we applied the calculated value in the experiments. We got very well results in this way.

http://www6.appliedbiosystems.com/support/techtools/calc/

Best regards.

Dear Arwa, both Tm and Ta of primers are very often misunderstood. Apart from the above discussion and knowledge input for your query I would like to impart here some more basic info, which one should know when dealing with the primers.

You, till this point already have the idea regarding primer Tm and Ta as per their normal  definitions. 

Now, what you must know that a primer's Ta should always kept less than its Tm. So lets assume in reverse that for a primer you have Tm less than the Ta, so every time when this temperature will reach your primers will always be in melted stage and there will be no annealing to the template at all, as the Ta is still higher than the Tm. So its very simple and logical without complexities. There will be no amplification or if anyhow there is the product, the yield would be negligible. For example if Tm is 60 C and Ta is 65 C so how come your primer will anneal to the template. I hope you got it.

A perfect ct curve will only appear if your designed primers are specific in terms of their sequences, Ta and the Tm. One of the major cause of double peaks is also poorly designed primers, though there are several other factors too are involved for multiple peaks.

In general as per primer guidelines, a primer Ta must be 3 - 5 C below its Tm is excellent no doubt.

Ta and Tm both are directly proportional to the primer length. Ta above 72 C not recommended at all and also a primer pair with a very low Ta may anneal non-specifically giving false products not the actual ones.

And important to know for the primer Tm, that both the F and R primers if are not having the same Tm (which is also fine), so their Tm difference should not cross above 5 C, as this will cause unequal extensions and often a failed amplification. Example, if F primer Tm is 55 C so R primer Tm by no means should cross 60 C. It must lie within the 5 C range. Ideal primer Tm by guidelines is 52 C - 63 C.

Highly rich  GC % also not good for the primers, as such primers cannot denature easily even upon reaching their Tm, so you will definitely face issues in your melt curve appearance, which are incomprehensible and nobody wants that. So follow the ideal GC ratio range, which is usually from 40 - 60% GC.

I do hope your concepts are more clear now. 

Sincerely.....

Above answers perfectly sum the things up. 

However, for me i cross check my primers on Primer-Blast and get the idea of TM (TM calculation methods/formulas are different for every software). Suppose TM of F primer is 60 and TM of reverse primer is 59. In my set up, my rule of thumb will be, take the lower TM  primer (which is 59) and subtract 2C from it so I will use 57C. That is how I begin my setup.

Regards 

Thank you, Mert Sudagidan, I've already calculated the Tm but my problem is the annealing temp. how can I choose it based on the Tm?

Thank you, Jawwad Ahmad, you are always answering my questions and I like your answers and all your valuable information you've got me. I got all your points clearly and my primers all (F and R) have the same Tm. and thank you again for writing all these concepts. 

Thank you, Ali Zohaib, yes, I used to do that too but I've found a paper used the same primer as mine and written they have used the annealing temp. at 60 but it's not working with me, I'll try that again.

Hi, why not you try gradient temperature?? try 5 degree temperature variation range from you Tm. Ideally the Ta is bellow Tm but not too low. It is a easy way to find the proper Tm for your primer. You may get more than one temp. with a single peak but you may choose the best one with highest intensity.

Moreover, the PCR reaction conditions mentioned in the published paper do not work all the time even the primer is same. It may happen depending on many conditions related to your environment, instrument, mastermix etc. It happend to me as well. So, anyway you have to optimize the PCR reaction and the reaction conditions yes of course the Tm.

Lastly, if you still have problem with your primer, then you can try to buy bioinformatically pre-validate primers from Qiagen. They works well and requires less optimization. you can go to their website (Gene Globe) and search your gene and then order.

Thanks and good luck