Please note that if you are interested in carrying out PCR reactions, the Tm is irrelevant. What matters is the Ta (annealing temperature), which you need to optimise. All the programmes used for calculating Tms/Tas will give you different results. The best way is to empirically determine the optimal Ta by using a gradient thermal cycler such as the BioRad CFX or by running separate PCR assays over a temperature range. The actual range depends on your primer set(s) but if your consensus in silico Tm is 62C, then try your PCRs between 54C and 60C.

I use PerlPrimer by Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 2004 20(15):2471-2472

You will find the method how it works in the paper

The opens source software can downloaded from: http://perlprimer.sourceforge.net/

The prediction of the melting temperature is tricky for longer fragments. The paper A Phenomenological Model for Predicting Melting Temperatures of DNA Sequences

Khandelwal G, Bhyravabhotla J (2010) A Phenomenological Model for Predicting Melting Temperatures of DNA Sequences. PLoS ONE 5(8): e12433. doi:10.1371/journal.pone.0012433

might be worth a read

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0012433

I use OligoExplorer (version 1.2) which is a free software download from http://www.genelink.com/tools/gl-oe.asp that I use to design primers (link is for most recent version 1.4). Just upload any text file containing your DNA sequence and the software automatically outputs the common parameters such as melting temperature and GC% of your selected stretch of sequence. Good luck!

You can use the Oligo Calc: Oligonucleotide Properties Calculator:

http://www.basic.northwestern.edu/biotools/oligocalc.html

Dear Andrew,

The melting temperature (Tm) of the DNA means "the temperature at which the half of the Double Stranded DNA becomes Single Strand DNA" means this is the temperature at which the DNA is denaturating or melting from its Native form, hence we call that temperature as melting temperature of the DNA. Now if DNA contains more GC base pairs( more hydrogen bonds comparitively than AT) then it requires more energy (temperature) for breaking the bonds. Here in this case the Tm of Primere varies depends on the composition. formula for predicting the Tm of the primer (4(G+C) + 2(A+T))-5.

Try it.

All the Best.

Thank you to everyone who has replied for these very helpful and comprehensive answers! All the best, Andrew.

it is possible easily by some bio informatic software from http://www.molbiol-tools.ca/PCR.htm and some other that guys said

also you can calculate based on percentage of G,C.T and A as Sumaiya said befor

good luck;)

About melting and how it programming(a few, as this part of work perform by IT ) you can see in my article "Changes in the nucleotide composition and pattern of DNA methylation during germination of cereals seeds", Minasbekyan L.A. ..., in Russian journal of Plant Physiology , 2000. vol.47, No.2,P.256-259. I hope it will helpfull. Good luck.

You can calculate online tolls like oligo calc, oligo calculator.

This will provide no. Of parameter.

You can try this link; https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator

but take into account that there are several methods to calculate the Tm and each formula gives you a different Tm.In oligo7 software you can see the Tm calculated from different ways and choose which you want.

Please note that if you are interested in carrying out PCR reactions, the Tm is irrelevant. What matters is the Ta (annealing temperature), which you need to optimise. All the programmes used for calculating Tms/Tas will give you different results. The best way is to empirically determine the optimal Ta by using a gradient thermal cycler such as the BioRad CFX or by running separate PCR assays over a temperature range. The actual range depends on your primer set(s) but if your consensus in silico Tm is 62C, then try your PCRs between 54C and 60C.

Dear Andrew

You can go to the following URL, insert your DNA sequence and find many information about it:

www.basic.northwestern.edu/biotools/OligoCalc.html

Thank you all again for the further helpful answers.

@Stephen Bustin: My interest here is in designing primers for LAMP rather than PCR. It's early days, but papers so far seem to specify Tm is very relevant for this one.

@Andrew Whiteoak: Stephen Bustin is right in general (here is the literature on this topic http://www.ncbi.nlm.nih.gov/pubmed/2243783). Most authors do not know the difference between the Tm and the Ta. With LAMP however you are also right. I covered this in part on one of my book chapters (unfortunately in German only). But keep in mind, all Tm are just predictions.

Are you aware of LAVA (http://www.biomedcentral.com/1471-2105/12/240)?

Check "GC-Profile" which might help you to spot visually GC reach areas of a sequence (you have to fiddle with the settings). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1538862/

Appart from that I recommend Unipro UGENE (http://ugene.unipro.ru/index.html) which is quite a nice tool to learn something about and manage your sequences. http://bioinformatics.oxfordjournals.org/content/early/2012/02/24/bioinformatics.bts091

BTW, did you check if there are tools provided by EIKEN?

Did you check this: http://primerexplorer.jp/e/ ?

Thank you so much!

No, I was not aware of much of the information above. I am very grateful and will be working through every paper mentioned. I am currently reading up ahead of my masters' start in October, and this is an entirely new field for me.