Hello everyone,
I am having an issue with my LAMP reaction for target DNA detection (30ng). I was using the standard NEB protocol using BST 2.0 polymerase, with 1.6 uM FIP/BIP, 0.2 uM F3/B3 and 0.4 uM LF/LB (final concentration) primers, MgSO4 8mM and I didn't use any betaine initially. It was working fine for 3-4 experiments with a good ladder-like pattern in positive tubes. It didn't show any ladder-like or single band in negative controls. but after 3-4 experiments, It started showing intense multiple bands in the gel images. I also changed the workplaces, reagents, and even a fresh set of primers but it is still showing the same false positive reaction in the negative test control. I also tried different concentrations of betaine (0.5 to 1.5M) and DMSO (2.5 to 7.5%), and lower primer concentrations also, but it didn't work. it's still showing amplification in negatives. I am not sure what is the problem I would be grateful for any valuable suggestion.
Thanks and regards,
Ankur
You have post amplification contamination in your system. Initially I would order ALL new reagents and plasticware including the water and move to another researchers work area and set up a run with 4 negative controls ( no template) and 1 sample using their pipettes at all stages and their plasticware. While doinf this strip down your pipettes and clean them all thoroughly with soapy water and wipe down your working area with soapy water and change your lab coat. If you get a negative reaction in another area then try moving back to your area and running more NTCs with other pipettes and if this works then finally set up another run with your pipettes. Use filter tips at all reagent preparation and sample setup stages
Hello,
I had the same problem. It is most likely contamination. LAMP produces a large quantity of amplicons, more than in traditional PCR. You have to be very careful, almost paranoid when working with LAMP. When I had this problem, I ordered new reagents but still had the contamination, which lead me to the conclusion my work space was contaminated (which was correct). I would suggest first try to do your reactions in a different work space and in the future do not open PCR tubes with LAMP products in the same space you use to prepare the reaction mixtures. It is best to have a different sterile space in a different room for each step of the protocol. If it does not solve your problem you will need do decontaminate your spaces, clean your pipettes etc. and order all new reagents.
Decontamination of the working benches, plastic wares, pipettes is the best option. Since, the basic trouble shoots were covered by you, this is the only factor which has to be ruled out. Further, better use filtered tip (if not) and use dedicated pipettes for addition of template and reagents
Dear Paul Rutland Kristýna Myslínová Narayanan Kalyanaraman, Thank you all. My experiment is working now. It was contamination, I washed all pipettes and devices properly. I also changed my Biosafety cabinet. and I ordered everything fresh. Thank you all for your valuable response. Ankur
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