| | Re: (LAMP) Problems with Loop-mediated isothermal amplification
so it seems to be a general problem. Everything indicates that it is kind of contamination. During LAMP huge amount of DNA is amplified and also the contamination.
The result of my experiments are all positive. On Gel, you can see typical ladder-like structure, for positive and negative.
The same in Real-time LAMP with FAM channel. Positive and negative getting positive after 25 to 35 minutes, the negative few minutes later. Melting curve analysis with sybr green indicates a wide peak between 55 and 70 °C and one peak at 89°C. The smallest product in LAMP is the dumbell-structure (starting point for exponential amplification) The calculation of melting temperature of this target region also showed 85 to 89 °C. The LAMP seems to work well but there is some contamination.
I changed LAMP reaction mix.
So it might be possible that the contamination is in BST-Pol or in Primers. But PCR results are okay. I have no idea yet.
I also tested the LAMP without one of the inner primers. When one or two of the inner primers are missing, LAMP is still working. When inner Primers are missing, LAMP doesn't work. This is logical because inner primers are forming dumbell-structure.
Does everyone solved the problem so far?