There exists several algorithms to estimate the Tm for primers, therefore you will get different Tm for the same primer from different sources.

Have you tried a temperature gradient for annealing to find the optimal annealing temperature for your primers? Because the additional band is smaller you can´t play with the time. If you don´t find a better temperature you have do design new primers.

Are those 2 bands same intensity on the gel? Could you post the picture for us to see? You probably can try a gradient PCR to see...

Hi, are you sure that you have no contamination? Did you sequenced and blasted your 300bp-band? But as Yuan-Yeu Yau mentioned: Try a gradient PCR! Cheers, Nadine

If you are doing RT-PCR, the second band may be a splice variant. Check gene databases whether this might be the case and, as suggested by others, sequence your PCR products. To do so, either cut out the band(s) from the gel and purify them using a gel extraction kit or clone them.

Thank you all for your advice. I have attached the gel picture. The first column is the ladder. The second column is the sample of interest; and I want the upper brighter band, not the lower one. I have cut and purified the lower band there seems to be no problem with multiple bands when I run PCR (see middle band b). However, if I run a PCR of the purified upper band in the column of interest, I continue to get the lower band. Ignore the last column - it is another sample

Even if you cut out band form a gel and purify it, there are almost always  traces of the other amplification products left in your sample. This is the reason why you obtain your two bands even after gel extracting the larger band. Since smaller bands are preferentially amplified you might not obtain two bands if you run your PCR on the purified smaller band.

Have you tried a Touchdown? Cheers. Sandra 

If you run a PCR using the purified upper band and you continue to get the lower band (as you stated). It might indicate that at least one of your primers actually anneal or partially anneal to somewhere inside the gene-of-your-interest. Have you checked (or BLASTed) the 2 primers to the whole sequence of the gene of your interest to see if any potential locations inside the gene where the primers can anneal to.

To optimize your PCR recation for "specificity", you should use higher annealing temperatures; perhaps you may increase the amplification period and decrease your primer concentration as well, but it may help to try just higher annealing temperatures first.

The common way to solve a problem with non-specific product is to increase annealing temp. About theoretical annealing temp: there are some factors that affect it: salt concentration and calculation model for example. Nothing gives you absolutely correct data. Perform Real-Time reaction in different conditions (annealing temperature) and analyze melting curve. Your product should have only one peak. Additional peaks corresponds to primer secondary structures, dimeres and non-specific products. 

To determine the hybridization's temperature of a primer, I think that we must first work in the temperature range given by the manufacturer and then gradually we dertermine the hybridization's temperature when you observe only one band of DNA on the gel revelation using specific primer. Also I think that can review the concentration of MgCl2. and then you must gradually reduce the concentration of this  salt. which concentration you are working?

everything is said about the experimental determination of the melting temperature.

If you use another taq or taq buffer, this may increase the melt temp up to 10 °C ... (for instance for a cheap and the phusion proofreading taq). Imho this originates mainly from MgCl2 concentration, but also other additives like BSA may have an effect. This might be a secret of the manufacturer. 

Keep in mind that there might be two bands every time with your samples depending on your genotype if the primers were not designed for this special genotype. The primers may give a single band in another genotype for which it was designed.

If this is the case you might have:

     1. an allelic variant with a DIP polymorphism in it.

     2. similar genes that are amplified. So the primers are not gene specific in this genotype.

It all boils down to which algorithm you believe in.  Algorithms forecasting Tm are still evolving...

Dear Mr. Karavina,

Please check the link below.. Maybe, it can help you.

Best regards,

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

gradient PCR

You can try the following things--

• Try Gradient PCR to determine the appropriate Tm. In most cases, the Tm provided by the manufacturer doesn't work.
• Try varying the MgCl2 concentration. In your case, you should probably decrease it  to decrease the specificity.
• You can also try enhancers like betaine, DMSO, PEG, etc.
• You can also try using a high fidelity polymerase enzyme.

Which is the template of your PCR reaction?

 As suggested by others: Do first a gradient PCR. If no suitable temperature is foun, try to use the QIAGENE Q-solution from the Multiplex pCR kit. Otherwise shift your primers a few bases to either direction (avoiding secondary structures, in particular at the 3' end.), (Delta G should be higher than -4)

You can please follow the links below to obtain certain crucial informations:-

1. http://nar.oxfordjournals.org/content/18/21/6409.short

2. https://www.lifetechnologies.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html