I am trying to validate the detection of plant fungal pathogen through LAMP reaction. But I have an issue with the LAMP reaction. I am detecting the band in NTC (Control) also.
To rectify the problem, I replaced all the reagents except Bst polymerase with fresh vials. MgSO4 concentration also reduced 6 μM, 4 μM and 2 μM, temperature also verified on 65 oC, 68 oC and 72 oC.
But still same results. Please suggest, how I resolve this problem.
LAMP Reagents Volume (for 25 μl Rxn)
10x Isothermal buffer 2.5 μl
Betaine (5M) 4.5 μl
MgSO4 (50 mM) 3 μl
dNTPS (10 mM) 3.5 μl
Primer F3 (0.2 μM) 1 μl
Primer B3 (0.2 μM) 1 μl
Primer FIP (0.4 μM) 1 μl
Primer BIP (0.8 μM) 1 μl
Primer Loop-F (0.4 μM) 1 μl
Primer Loop-R (0.4 μM) 1 μl
Bst Polymerase (8U) 1 μl
Nucl. Free water 4.5 μl
DNA (10 ng) 1 μl
Those are likely a result of misamplification, not a contamination. Basically there are two ways to resolve:
1. Use shorter duration for your assay (ie use maximum duration that results in no amplification from NTC)
2. Redesign your primers/probes
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