I'm trying to collect microsatellite data using primers developed on a related species but instead of having primers with fluorescent dye already on them, I'm trying to attach an M13-dye to the forward primer which also has an M13 tag on its 5' end. I am having some difficulties though because my negative control bands look identical to the positive bands, regardless of annealing temperature and MgCl2 concentration, and the bands seem too large for it to be primer-dimers, it seems like genomic DNA.. it's about 100-150bp.. so I'm not sure what is going on. Before somebody suggests that it is contamination - I started over at a whole different lab bench, different pipettes, pipette tips, reagents, ordered new primers, thermocycler, everything, but the problem did not go away. Since I'm still optimizing the microsatellite primers I also ran a test using some invertebrate COI which I have positive results from in the past, and included a little bit of each reagent into each reaction instead of DNA, and I got a very strong band at the M13-dye. This was brand new, reordered after the last batch had this problem, suspended with TE and diluted with nuclease-free water. I am wondering if it has to do with the gel sybr-safe filter? Or the UV illumination for visualizing the gel? I am grasping at straws, please help or let me know if you have the same problem. The dye is just M13 labeled 6FAM: 5'-/56-FAM/TGTAAAACGACGGCCAGT-3' from IDT

  • 1
    $\begingroup$ I have no experience with this, but what is your negative control? $\endgroup$
    – canadianer
    Feb 22 '17 at 1:06

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.