I recently started working with LAMP (Loop-mediated Isothermal Amplification) using target DNA. I designed 4 primers using PrimerExplorer V3. I use LAMP Kit by Lucigen, Inc. For some reason I do not see any turbidity in the tubes after 1 hour of reaction at 70 degree C in the thermal cycler. On the agarose gel, I can see multiple bands for my LAMP reaction products but I also see huge smears throughout the lanes along with thick band near the gel. My negative control (reaction mixture without template) gives me the same result. Can you suggest what is wrong with my protocol? Thank you for your help.
Like any PCR based assays, LAMP is also prone for false positives (bands in negative control) due to template contamination of regents.
The following link has good information about how to avoid contamination in PCRs. Same principles apply for LAMP
http://bitesizebio.com/20773/clean-up-your-act-how-to-clean-up-pcr-contamination/
Hi,
Just like Panduranga said, it is most likely an amplification artifact in your no template controls. Try decreasing incubation time, but if you dont see specific products at all, it might be something with the oligo design/template cc. Here is a general guideline:
https://www.neb.com/protocols/2014/11/21/typical-lamp-protocol-m0275
Sometimes, maybe due to contamination. I also faced the same problem.
Can I know the volume of MgSO4 that have been added in the LAMP reaction? excessive amount of MgSO4 also can cause non-specific amplification.
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