I've tried many web based apps available online to calculate the Tm and each one of them were producing completely distinct results which had caused me so much confusion. I've got 5 sets of primers, 10 in total and all I want is to find the Tm, and annealing temperature of each, so I can set a temperature gradient (+/- 5C) as suggested by many to do the initial optimisation. Please suggest me the easiest way where I can submit my primer sequences and it'll generate the correct melting and/or annealing temperature for each set.
It depends on what type of media interface you require... ie. Contact measurement or contact-less measurement.
If it is contact measurement then , Suggest you use Pyrometer with a replaceable tip... we use for measuring the molten metal temperature in foundry.
In case of contact less measurement then air ambient measurement devices such as Thermometers only would be suitable (duration of exposure of the probe depends on temperature and the enclosure material characteristics.
Hi, the company who sold you the primers also sent you a datasheet with all relevant informations. Why to calculate the melting temperature again? Cherrs, Nadine
The melting temperature is given by the sofware used to design the primers. If you "find" these primers in an article, you could "test" them in primer-blast. This software give you a calculated Tm and the size of the PCR fragment if it find them in the genome/refseq.
For the annealing temperature, you will find it in the booklet of your enzyme. It depends only of the taq polymerase (often between 68 and 72°C).
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
If you are doing real-time PCR and have access to a machine that does a melt profile there is one failsafe way of determining the melting /annealing temp of primers. Buy complementary sets of primers (smallest synthesis scale). Mix each of your primers and its complementary primer in equal molar amts in the PCR buffer that you intend to use (at the concn normally in PCR mix or up to 5x , along with Sybr Green) and do a melting curve analysis from 50C-80C (for example). You will generate a melting curve peak at which 50% of the primer sequences are s/s. The max annealing temperature will usually be about 5C below the Tm -just before the peak starts to rise.
There is no accurate algorithm to calculate Tms of oligos as they are dependant upon the salt and Mg concn in the buffer in which you do your reaction - the Tm will be different for different PCR master mixes (in my experience up to 5C variation). if you are going to be doing multiple expts with the same primers this is a very cost effective way of getting it right in the first place.
If you do not have access to a real-time PCR machine, I have found that for my primers the "nearest neighbour" calculation is the best approximation, but as with all the algorithms it is primer length dependant. Trial and error with a temp gradient will get you there - use a calculated Tm (usually supplied with primer) and do a range +/- 5C. Calculated Tms do not necessarily reflect the true Tm if there are internal sequences of secondary structure.
Ian
Hello, Harindra, you can use the following link by placing the sequences and selecting the type of polymerase you are going to use. good luck
https://www.thermofisher.com/pt/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html#/legacy=www.thermoscientificbio.com
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