Hi,

I have been trying to amplify a segment containing my gene of interest in the TOPO TA vector pCR2.1 using the sequencing primers M13 and M13(-20). However, it seems that I am getting some additional bands at +/- 3000 kb that don't make any sense even after modifying several parameters such Tm, extension time, template amount etc etc. My genes of interest 9(5) and 51(2) are 1.35 and 1.75 kb long approximately, and after adding the extra length due to the position of the M13 primers, the size should be around 9(5) --> 1.5 kb and 51(2) --> 2.0 kb. These bands show up in the gel as you can see. However, there are some additional bands close to 3.0 kb which I cannot understand.

I should mention that I run a PCR using only specific gene primers and they worked well. In theory there shouldn't be any other annealing sites for the M13 primers in the pCR2.1 vector, so I don't know why I am getting the extra bands??

Has someone faced a similar problem in the past?

Any advice on how should I proceed? I am trying to get my genes sequenced and that's why inserted them into the vector and try to amplify them using flanking sequences, so that I can get my whole gene sequenced and I don't lose the first 50 bases or so.