I am doing a PCR from DNA extracted from blood (human) and bisulfite converted. It used to work fine until a month ago when the negative control (water + buffer+ Mg+dNTPs+ primers+ Taq platinum) started showing a band at the same size of my expected product (255 pb). It is a faint band and not always appears (about 50% of my PCR show band in the negative control). My annealing temperature is 57°

I changed all reagents (Taq also, same brand and same lot) and buy primers twice, I tried different things (a different person did the reactions also…many things).

I am also doing a PCR for the same region, larger amplicon (549 pb) but for the DNA without bisulfite treatment and the only difference between the two PCR are the primers (annealing temperature 62.5°), all the other reagents and PCR conditions are exactly the same. Before, I had contamination in this reaction also but it solved after buying new primers. Now I don´t have any contamination in the negative control of this PCR.

Both pair of primers were bought at the same time and diluted with the same water and at the same time.

I know I should not see anything in my negative control but I am running out of time. I was wondering if it is possible this: If I sequence (Sanger) my pcr products and also my negative control and I don’t see anything in the negative control, can I trust in the sequence that I get from my sample? I guess I can…

I am attaching some photos. The first one is a negative control and my sample, this negative control was ok in my opinion. Next day same PCR: first one is the negative control and four samples. Sometimes the faint band is not seen in photos.