I have experienced some weird results from my sequencing lately.
I'm trying to sequence inserts in pCR2.1-Topo vector using the vector primers supplied within the kit (M13 forward (-20) and M13 reverse).
Prior to growing Over Night (ON) cultures on the clones, I test using the vector primers that the clone indeed does contain an insert. This PCR works fine and I can pick the clones that appear to have an insert of correct size. (The fragments a want to clone, may vary in size and I need a clone in a certain size).
After purifying plasmid from ON cultures, I sequence the clones to pick a clone with the correct sequence. I have done this many times before, but not by using the vector primers. Here is the problem:
1. It appears that the company has mixed up the primers, because I get the reverse primer's sequence with the forward primer. This I can see because after a few hundred bases the forward primer's sequence pops up. If it really was the forward primer that was used it should have been the reverse primer's sequence popping up. FYI it is not me mixing up the primers, I have done the experiment twice Has anyone else experienced this mix up?
2. One of the primers NEVER work in the DNA sequencing. Taken into account the potential primer "mix-up" it is the M13 Forward primer that never works. This is strange to me since the to primers as a pair function fine in the PCR. Does anyone have an explanation to why the primer is not working when used for DNA sequencing?
I have attached the chromatograms for both primers for one of the clones, so that you can see how it looks when I don't get a sequence for the forward primer.
Thanks in advance