it's still the same problem. When running LAMP without dNTP, no reaction takes place. Normal LAMP results in false positive in NTC. I also changed concentrations of betaine, mgso and results are the same. I made all buffers new...still same.
So i think the primers are contaminated somehow or the primers are hybridizing and amplifing. But in case of self-annealing and amplification, is it possible to get ladder-like band pattern on gel?
Maybe the long primers (FIP/ BIP) are very sensible because they are much longer than normal.
Primer redesign never solved problem. In case of contamination, how to get rid of it? Room separation for Mastermix, reaction and detection is done?!
Originally Posted by msarah
Have you solved your problem yet. We are running two different primer sets for completely different target genes. One consistently gives us good results and the other has false positive. The Bst pol is the same for both runs. I am beginning to think that the primers might be reacting with each other in some way. We use the lower concentration of Betaine 0.8 M which increases the stringency. The next step in our plan is the move the primers slightly and check very carefully that there is absolutely no change that they are annealing to each other and giving the false positive. As far as Acinaria having + results with no primer present is very strange. We had positive results when no DNA template was present , but - results when BST or dNTPs were not present. this leads me to believe that the primers are reacting with each other and have to be replanned.