Sequence is just the same as I ordered or used earlier but the Tm is different. Tm that was ordered by me was 51 and 52 Tm of primers I have received has 69 and 52. Is it possible if the primers having same sequence and number of bases but having different Tm ? If it is so then do I need to optimize my PCR conditions again?
Dear Vipasha,
Firstly, do the PCR by applying same parameters as applied in previous one. Observe the result. I think you will get desired band. If not then go for optimization.
Yes, I am agree with Dr. Kumar. You can follow the suggestions given by him. Hopefully, these will help your research.
The platform you used to calculate the Tm is rpobably different from this that each company or laboratory uses. So don't be suprised if the Tm of your primers is quite different. I always calculate the Tm with one platform, and set up my experiments according to this. I never take care about the tm that the company gives. For Primers that I ordred more than one, and from different companies (which mean different Tm), I worked with the first parameters and I got good results.
You Just need to try a PCR and see..Good Luck
Dear Vipasha, you have nothing to worry about if your sequence is same and if you had already standardized your previous primers. As many authors said in this page, I agree with them in the way that, there are different methods for Tm calculation. That is the reason why yours is different now.
Just try and give a feedback because I am very interested to know what will happen.
All the best in your experiment.
Hi, Artur is right:
An oligo has a sequence-dependent melting temperature, hence if you don't change the sequence and/or the chemistry of your oligo the Tm will stay the same.
The difference between the Tm values can be explained by either using different algorithms (Nearest-Neighboor, salt-adjusted or a simpler model) or the parameters used for the calculation: salt concentration, oligo concentration and the like.
Same primer sequence same Tm. Try them in a PCR, they will work, and let us know the result.
good luck!
Although different methods of calculation do lead to different Tms these differ by only a few degrees. Something is wrong if you calculated 51C and the suppliers calculated 69C. In reality Dr Saera-Vila is correct - any primer sequence can have only one Tm in a given set of ionic conditions. So in designing your primer, have you added a sequence that doesn't hybridise to your substrate and you did not count in the calculated Tm, such as a restriction site or sites, an IRES, a promoter or similar. The supplier will assume that these regions are part of the Tm calculation: and you should remember that after the first cycle of the PCR the Tm for this primer will be the higher figure, so there may be a slightly higher percentage of off-target products if you are using a hybridisation temperature around 50.
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