Thanks so much. i was thinking the same too and not sure until someone advised me to do so. The primers were obtained from the literature and difference between forward tm and reverse tm was about 3C. not sure which range of temperature gradient should i choose. I have read we need to choose the lower temperature than tm of primers. is it right?

thanks

What is the size of the smearing band? If it is below 100bp, then you just see your primers. Note, that in 30% primers from literature contain mistakes, as well as wrong orientation. Be sure, that they are correct. Tm 60 is usually ok, but you can start from 55 and 30 sec of elongation. In most cases this results in PCR product with small nonspecific bands, which you can get rid off by increasing the Tm. Good luck!

Tm is given by a general formula 2*(A+T)+4*(G+C). But practically it depends on other factors as well. So correct Tm is given by the company from whom you got your primers designed. If you could not get from company you can calculate by the formula only.

After getting Tm, put Plus(+5) and (-5) degree to Tm as gradient, cox annealing temp is +or-5degree of Tm

The size of the band is 314bp. I just got very faint band after i diluted the PCR sample with water (1ul in 19ul water). 20ul is the right volume as stated in the leaflet of e gel.

I will try using gradient temperature of annealing step and also other genes as well.

May i know which number should we add in this formula: 2*(A+T)+4*(G+C) to get the annealing temperature? Are those the number of A or T of the primers? May i know the reference used in such formula?

Thanks

I do not understand why you need to dilute PCR amplified products. I have not heard it before.

Please check the link for reference of Tm

 https://www.researchgate.net/post/Formula_for_calculating_Tm_of_primers2