Hi, this is my first post in molecularstation.
I am trying to PCR amplify a 1.5kb sequence from a pET28a plasmid vector containing my gene of interest. The first time I tried about a month ago, it worked fine. Please see attached picture.
However, when I tried again with the same plasmid (50-100ng), primers (10uM), buffers, and NEB phusion polymerase a few days ago, there was a smear tailing my PCR product. See other attached picture.
I grew the plasmid-bearing E coli cells in a drug media and miniprepped it, and I diluted the 100uM primers to 10uM and used those as well. But the smear appeared again.
All PCR reactions were 50ul and I loaded 10ul to each well. I suspect that the my primers, which I ordered a month ago, became degraded, or my plasmids went "bad" over the one-month period.
Has anyone else experienced this problem before and how may I fix this problem? Thanks