Dear Marina,

After you have checked their specificity using NCBI blast the next step is to order them and confirm their specificity by performing PCR amplification and then checking the PCR products with gel electrophoresis. If there is only one band at the size range you are expecting then the primers are specific. In that case you can also perform Sanger sequencing on the PCR product in order to confirm that the product is the one you were supposed to amplify.

Try to blast the sequence of your primer and probe vs all the sequence are deposed in the NCBI database.

The other option is only to try them in qPCR (especially by the qPCR you have the confirmation of the primer and probe).

Dear Marina,

I agree with Constantina and Giorgia. For me the best way to be sure: Sanger sequencing. I always do it after reaction optimization. But sometimes it is quite dificult to receive sequence of good quality because of short amplicon. In that cases I use cloning (dT).

Moreover, my own experience tell me that in 99% (with TaqMan) you receive the right reaction product. And it is enough. However in the most important works it is preferable to receive an exact sequence by Sanger.

Andrei S. Babenka thanks for your answer, could you send me a good protocol using cloning as my amplicons are short

I like TOPO TA cloning kit

https://www.thermofisher.com/order/catalog/product/450641?SID=srch-srp-450641

You can use manufacteurer's protocol. But:

1. You need competent cells (bacterial culture for example DH5a E. coli)

2. Specific primers for TOPO vector.

Better one could be TOPO™ TA Cloning™ Kit for Sequencing. It is without competent cells, but optimized for sequencing with specific primers to TOPO Vector. Primers are included.

https://www.thermofisher.com/order/catalog/product/450030?SID=srch-srp-450030