Hi Slavka,

You could try a touch-down PCR. It is a modification of the temperature gradient. The initial annealing temperature is set about 5-10°C above the optimal annealing temperature. Afterwards, the annealing temperature is decreased every other cycle by 1 °C. After these touch-down cycles the normal PCR program is continued. This method increases the amount of your desired product in the first cycles. Therefore, the final amount of your desired product should be increased in comparison to all other formed products.

Good luck with your PCR!

Dear Slavka

It is obvious your PCR is not working well. If you expect a shorter product and still get the longer primary PCR porduct, it probably means your inner primer is attaching to the outer primer used during your first reaction.

Make sure your two primers are different enough. Clean your PCR products to ensure the second PCR conditions are appropriate (amount of DNA, salts concentrations, etc.)....

Just some thoughts

Pablo

If you still see the product of your primary round of PCR after seminested PCR you may add too much of PCR product 1 to PCR round 2! The amplicon of round 1 should serve as template for the primers in the seminested reaction. Therefore, only traces of the first reaction should be used as template for the second PCR. The larger the volume you transfer from PCR 1 to PCR 2, the higher the portion of primers from round 1 competing with those of round 2. So, my first hind would be to check, how much volume you need as template in your seminested reaction.

Next, make sure that your PCR conditions are well balanced. The seminested primer must fit with its annealing temperature and physicochemical parameters to the outer primer. Also, check that your seminested primer is of correct sequence and orientation. Good luck and best regards.

Thank you for your advices.

Probably, the amount of PCR product would not be problem, because I have used only inner primers in single PCR and no product was seen.

Primers are correct, I didn't design my own, I use primers from references.

So I will try to optimalize my PCR.