Originally Posted by sittingchair
HNB at a final concentration of 120 uM is consistently reported to have no inhibiting effect on the LAMP assay. You should try to repeat the assay with this concentration doing two variations of a MgSO4 gradient: one with HNB at 120 uM and one without. Prepare the master mix for both experiments together, then divide it out.
I'm also working on a LAMP assay and here is my problem: no ladder banding has been observed so far, however I am picking up a single band at around 4.5k in all of my positives.
I blasted all combinations of primers on NCBI and found that a potential product of the F1c and B1c ends of FIP and BIP respectively could produce a product of 4.5k in length.
Any ideas of what could be going on? Is it possible that my primer ratio (1:4 outer:inner) is simply not optimized?
For me it workes most of the cases when I melt template and primers together and THEN add LAMP reagents, which is of course not favourable, but you can try at least
90°C for 5min, cool and add reagents
I am using the same Outer: Inner Primer ratio of 1:4.
Sometimes I get great ladder pattern, repeated another day it's just DNA smear on the gel