| | Re: (LAMP) Problems with Loop-mediated isothermal amplification
Hi, new person here.
I've just started working with an LA-500 LAMP device, and I'm the first person to use it in my lab. I'm a first year co-op student, so naturally the first assumption we're all making is that I've been sloppy with changing pipette tips and such, but as time passes that's looking less like a possibility.
We ran into some early issues where all results were coming out negative. This turned out to be an issue with insufficient vortexing of the buffer (we're using pre-mixed Thermopol Reaction Buffer from Lucigen), resulting in poor function of the Bst Pol. So, we fixed that problem.
Then I started getting false positives. After about a week and a half, we put aside the primers we had been using (for eae and stx2 genes) and started fresh with stx1 primers and all new reagents. At the same time, I began preparing master mix in a BSC, and aliquoting it and template into reaction tubes in a separate laminar flow hood used on for that purpose.
That was working great for about 3-4 days, then random false positives started popping up again. At this point we're back to full on contamination.
JMCK and jensf's advice makes a lot of sense to me. In the confusion, I've been checking my LAMP product on gels, and then putting those tubes back in the same -20 freezer as my reagents. Since I'm being so careful to prepare everything in hoods, douse everything in DNA-Away and make sure I'm not just mixing template into my sterile water, contamination of the reagents in this manner seems like one of the only options.
Aside from that, when it works, it works well.
Any other ideas about what could be wrong here?