Hi! Andrii,

There are many software's, if you Google you get hell lot of information. Do not worry about that.

So far what ever the primers I designed are using by Primer 3 (NCBI).

Submit the sequence, it automatically calculate Tm, do not worry how to calculate for that (you had standard formulas for all), just reduce 5 degrees to Tm then you get Ta and set PCR it works fine.

I designed many primers, they all worked, got very good results.

Try it. You can save lot of time. I am sure you get nice results.

Good Luck!!

Hi,

I am not sure if there is universal formula. I also met the problem that different sources give different values. But for standard applications you don't need very precise Tm value.

Now I am using : http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/ .

Tm also depends on your conditions: buffer type and concentration, salt concentration, additional agents (like DMSO). So surely it is not only about primer sequence.

I too agree that the Tm calculation depends on various factors like Mg+ and other buffer concentrations. I would suggest BioMath Calculators for Tm calculation.

(http://www.promega.com/techserv/tools/biomath/calc11.htm) and you can also use https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator

I have also used Biomath calculator.

Hi! Andrii,

There are many software's, if you Google you get hell lot of information. Do not worry about that.

So far what ever the primers I designed are using by Primer 3 (NCBI).

Submit the sequence, it automatically calculate Tm, do not worry how to calculate for that (you had standard formulas for all), just reduce 5 degrees to Tm then you get Ta and set PCR it works fine.

I designed many primers, they all worked, got very good results.

Try it. You can save lot of time. I am sure you get nice results.

Good Luck!!

For PCR and primer lengths of 18-25, the GC/AT-method is good enough:

Tm = 4*[C/G] + 2*[A/T]

The annealing temp should be ca. 5K lower.

This "method" is not at all precise, but to the best of my experience by far good enough for the purpose of PCR. Keep it simple!

If your aim is not PCR but some other assay that might sensitively depend on Tm's, you have, in principle, two possibilities, both of which are based on empiricism:

1) try different formulas and/or

2) try different primers

and see which ones work best in your assay.

The best way in my opinion, and if you have a PCR machine where you can set up a temperature gradient, is to try several temperature around the calculated annealing temperature.

This will allow you to precisely define the more efficient temperature.

you can use any method (there are a lot of free softwares on line). The advantage of these methods is that they represent an easy way to detect probable dimer primers and loops. Regarding the melting temperature, what I do is to be sure that both fw and rv primers have the same Ta, and then you can set up the reaction in your first PCR reactions.

The CG/AT method may give you poor results, I prefer any program...

I have always used eprimer3 (http://emboss.bioinformatics.nl/cgi-bin/emboss/eprimer3) and it works quite well (or any of the other EMBOSS tools). I Hope it is of any help.

Hi, exactly there are several ways for caculate Tm, such as 2+4 rule of thumb, Linear regression(Tm = 81.5 + 16.6(log10([Na+])) + .41(%GC) – 600/length), but a simple way is using of softwares. one of the best softwares that can calculate Tm is specific and degenerated primers is Oligo analyzer. Also this online software informed about self and hetro dimers too. here is the link:

http://eu.idtdna.com/analyzer/applications/oligoanalyzer/

I have investigated the best way of calculating Tm a few years ago. I took the pain to actually implement a calculation program for the best possible estimate and checking who on the Web produces a state-of-the-art estimate. I found that the only utility on the Web that really implemented the knowledge that has been published in Tm research literature was IDT's Oligo Analyzer. Every value I calculated for various oligos was exactly matched by the IDT Oligos result. But IDT Oligo Analyzer still needs improvements in its user friendliness. However, all the other Web sites are still using obsolete estimate methods. Those estimates can be very confusing and cause damages to a researcher, as discussed here.

http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

After knowing the Tm, I usually do gradient PCR in the range of + and - 5 degrees of Tm. Then you can adjust the Mg++ concentration (between 1 and 3 mM) for best results

Hi, here is the video I made: how to calculate a melting temperature of the primers http://www.youtube.com/watch?v=LGb6y4xn93w

I think that it is more important to check if both forward and reverse primers share the same Tm and have no interactions (to avoid primer dimers), that to know exactly the Tm. I also calculate the melting temperature but after that, I play with gradient temperature, Mg concentration and/or additives (DMSO, spermidine, etc).

For same primer, even I am getting different values of Tm using primer3 and oligo analyzer.. I don't know which one i should go for.. and which one is more reliable. bcz then only i will b able to decide upon Ta precisely.. to confirm the correct value of Tm when i went about checking it with some other source then it gave me a third different value.. so I am confused....

Hi!

the following answer I posted on March 2013 when Andrii asked. It is very clearly mentioned what to do. I wonder why u confuse!

Hi! Andrii,

There are many software's, if you Google you get hell lot of information. Do not worry about that.

So far what ever the primers I designed are using by Primer 3 (NCBI).

Submit the sequence, it automatically calculate Tm, do not worry how to calculate for that (you had standard formulas for all), just reduce 5 degrees to Tm then you get Ta and set PCR it works very fine.

I designed many primers, they all worked very fine, got very good results.

Try it. You can save lot of time. I am sure you get nice results.

Good Luck!

Hey Jetty..thanku so much for ur suggestion.. i read ur answer before also and i am getting ur point.. but my concern is that, is primer3 more reliable over netprimer and oligo analyzer??.. bcz all three are giving me different Tm values... if that is the case then i can proceed further with using primer3 only...

Hi Mandeep,

My earlier answer fits right to your question here.

By the way, if you want to find a reliable Tm estimate tool, you need to know a judgement standard. Comparing all the tools you find and compare their prediction accuracy using the judgement standard. There are two ways to this. One way is to use oligos of know Tm and plug them into the prediction tool to find out the differences. If you have enough known oligos you can arrive at a statistic value for that prediction tool. The other way is to use what has been published in the literature and compare the prediction tools with the literature methods. I did both ways, and found that IDT's Oligo Analyzer tool is the best anywhere.

With all that said, I have to remind you that all these estimates are estimates only because your reaction conditions are bound to be different from the assumed condition used by estimate tools. If you want to know the exact Tm of your oligos in your reactions, you can find them out using a good gradient PCR machine.

=======My earlier answer copied below=========

I have investigated the best way of calculating Tm a few years ago. I took the pain to actually implement a calculation program for the best possible estimate and checking who on the Web produces a state-of-the-art estimate. I found that the only utility on the Web that really implemented the knowledge that has been published in Tm research literature was IDT's Oligo Analyzer. Every value I calculated for various oligos was exactly matched by the IDT Oligos result. But IDT Oligo Analyzer still needs improvements in its user friendliness. However, all the other Web sites are still using obsolete estimate methods. Those estimates can be very confusing and cause damages to a researcher, as discussed here.

http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

Jetty Ramadevi, Yanglong Zhu, Saira Bano... Thanku so much for your valuable suggestions....I am working on primer designing, keeping ur suggestions in mind... will get back to you in case i encounter any problem..

@Saira Brano Do you mean NCBI's "Primer BLAST"? NCBI page says it is primer 3 based (quoted below). Therefore the Tm is only as good as Primer3 can be.

======http://www.ncbi.nlm.nih.gov/tools/primer-blast/=====

Primer-BLAST

The Primer-BLAST tool uses Primer3 to design PCR primers to a sequence template. The potential products are then automatically analyzed with a BLAST search against user specified databases, to check the specificity to the target intended.

@ Mandeep Kaur Tm is only a small part of the primer design. IDT provides a set of tools for the primer design work. However it lacks an integrated capability of searching for genome-wide repetition, which Primer-BLAST seems to excel.

thank you very much for your answers but i have the same problem of Andrii that the primer vial mentioned that the TM of my primer is 52.9 for both reverse and forward but when i calculate on Primer- BLAST the TM was different it was 63 and 62. and the annealing temperature must be below or above the TM of the primers and if i have no gradient thermalcycler how to calculate the TA

Hi!

You mentioned as per the vail it was 52.9 and by using Primer around 62. You told that you do not have gradient, you have to make trail once by setting at 58 or 57C taking 2, 3 samples. Later verify the bands by running the gel so you get an idea whether to increase or decrease the temp.

Only thing is you should have enough patience to do all these.

Good Luck!

thanks Jetty for your answer i will do it and i hope it work

the easiest way or starting point as Jetty suggested, jut minus 3-5 from the Tm and check on agarose gel for amplification/non-specific bands. If there are non-specific bands try the next temperature till u get clear bands on agarose. goodluck

for the Tm calculations, some programs ask you to complete the NaCl concentration, the oligo concentration, and those values really make difference.

check that, and also, you can check with a primer with a known sequence and Tannealling, one that you already use, and see which is the Ta that the program suggests... it is a bit empyrical, but it works in order to know which program to choose. Good Luck !!

IDT's Oligoanalyzer (https://www.idtdna.com/calc/analyzer) works very well. Requires login. I recommend setting the concentrations of dNTP and Mg++ that you will use before clicking "analyze". Do not forget to check dG (Gibbs free energy) for hairpin, self-dimers and heterodimers.