I am working on LAMP PCR. The problem is, that initially I was getting positive results for my samples but suddenly I am not getting any amplification in reaction tubes for the same DNA?
I do not understand what the actual problem is because each time I am using fresh reagents?
If it s not the reagents, then it s either your template DNA (different genotype, polimorphic to your amplicon's priming sites? possible degradation? [check on nanodrop or similar!]) or your primers degraded. I'm assuming here noone played around with your cycling conditions.
Are you able to specify the LAMP profile you are using? Just to clarify, I am presuming you just mean you are performing a LAMP assay and not doing a PCR of the LAMP products afterwards (i.e. LAMP is not PCR)?
Thanks Siddharth.
Ya, I ran the LAMP product on gel and it gave a characteristic ladder like pattern and also turbidity was visible in the reaction tube. I got no bands and turbidity with No Template Control.I got positive results initially, but now, I am not getting any amplification in the positive reaction tubes. Even the primers are also new and they do not get degrade so early.
i am using genomic DNA of one bacterium as template.
i didn't check my F3B3 set and just ran the lamp reaction using all primers(F3, B3, FIP ,BIP). I'll check them but jst wanna clarify, should I perform normal PCR (consisting of all 3 steps) in order to get the check them?
Yes I checked the quality of dna on nanodrop and also performed PCR....
I am using 2 sets of primers F3, B3, FIP and BIP. My outer primers are working fine amplifying the expected product, but not the FIP, BIP. I have titrated Mg2+ from 1.5 to 8mM, and temperature from 55C to 70 C and also primers (FIP, BIP) from 0.5uM to 2.5uM. Despite this my LAMP didn't give any positive amplification. Can someone please explain this problem?
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