I am conducting a LAMP assay, and while the negative control shows no bands as expected, my positive control samples consistently display only a single band instead of the anticipated ladder-like pattern. What could be causing this issue, and how can I adjust my protocol to achieve the characteristic ladder-like pattern in the positive samples? I've attached a photo for reference.
Im not sure but my adviser says the primers are not binding to the whole DNA and the whole DNA is retained at the upper part of the gel. We’re currently trying initial denaturation, you could research on it. But we still have no bands
in my opinion it can be cause either due to primers which are not designed as per the requirement (very high chances) or due conc. varieance of DNA. Make sure to incubate the adequately (according to me 70-80 minutes minimum).
Following points must be carefully incorporated if LAMP Assay is not giving you the desired observations
1) Most importantly check Mg ion come (around 5 nM still not sure) which will enhance the aplification process and the tempreature must be kept between 80-90 ideally for better results.
2) If the primer is not complementary to the LAMP sequence, the results will differ from what you actaully desired. In this design a new primer to ensure the perfect complimentarity in sync with the LAMP sequence.
3) Use of a positive control which have a sequence know to you.
The above mentioned steps is sufficient enough to ensure adequate amplification (use PCR if required) and can inhibit the formation of primer-dimer entity (increse the DNA concentration if necessary).
I guess these steps will make your procedure hastle free. Please let me know if I was able to cater your query aptly. However I performed LAMP assay in my B.Tech (Engineering) degree in Biotechnology with Specialization of Genetic Engineering in Functional Genomics module, which equipped me with an experience in Biotechnological Tools espicially primer design and generation of cDNA libraries to read and analyse sequenmces as per the situation and then carry our its apmlification (RPA or PCR, whichever needed accordingly). If you need some ppt slides, explaining the concepts and science underlying biotechnological tools working principle and its efficient use in experimental set-ups, I can mail you.
Regards,
Abhishek Anand
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