Hi everyone,
I have been facing problems with inconsistent results with LAMP. I ran LAMP on a lot of samples today, and included 3 blanks. One of the blanks amplified. Some samples that were meant to be negative also amplified. All my positive controls amplified properly though.
Previously I ran LAMP with the same protocol, reagents, primers, basically same everything on DNA templates other than the template with the target DNA, and some blanks, just to make sure none of my reagents or pipettes were contaminated. Nothing amplified then, which is good.
I make the LAMP mix in a different room, add template DNA in another room, and run the gel and add dye in a third room. LAMP products never come in contact with the first two rooms. I use filter tips for making the master mix and also for adding template DNA all the time. I always turn on a UV light on the work benches after I'm done with everything. I try to rule out contamination as much as I can, with like often changing gloves, opening the tube caps carefully when doing the gel and adding dye, etc.
I did notice a difference between the ladder pattern in positive and negative lane in the gel though. The ladder patterns between the positive control and negative is distinctly different (see picture below). Is it possible that it's my primers amplifying?
My primer sequence:
F3 GCTGGCGTGATCTCCTCTA
B3 TAAGTGTGCCGACTAGGG
FIP TCTGCCAAGACAAGGGTGCAT-GTGAGGTGCCAGATCTATGG
BIP GTGCAGTGGCGGAATCGTGG-GATCGCCAAACACACTGACA
My LAMP is based on a 25 ul reaction with 2.5 ul 10x Isothermal Buffer (BioLabs), 8mM MgSO4 (6mM + 2mM from the buffer), 1M Betaine, 1.4 mM dNTP mix (10mM each), 40uM of each FIP and BIP, 5 uM of each F3 and B3, and 8U Bst Polymerase (BioLabs), 2 ul template DNA, and molecular grade water.
If anyone has faced the same problems and or know the cause and solution, I would really appreciate some help. Please email me at [email protected]. Thanks!
Yes it can happen that you have amplification with negative samples or control without knowing where the problem come from. Few suggestions that may help:
- Keep everything on ice, reagents, master mix, and return them to the freezer as soon as you finish to prepare your Mix.
- Put your DNA in the tubes before the Mix, that can be done one or 2 days before and store the tubes so that you will not have to pipet DNA.
- Avoid also to put together DNA and control tubes when dispensing the Mix.
- vortex and spin down very shorty and the same way for any tube.
Hi Nam Vu and Athanase Badolo,
Thank you for your recommendations. I will try them and I hope my results will improve :)
Hi Maria
I have extensive experience of LAMP and I'm afraid looking at your gels that you are encountering low level contamination at the minute. The golden rule in our Lab is never open the tubes post amplification! This was learned the hard way. I would advise ceasing gel electrophoresis if at all possible as this is the most serious contamination risk. If you continue to encounter contamination I would discard all in use reagents and begin afresh. Utilise UV cabinets for mastermix production, QC each batch and run multiple negative controls to ensure contamination free. Good luck
Yes one you have established that your LAMP is functioning (Specificity) use visual detection eg. Calcein, HNB or turbidity. Can also employ intercalating dyes eg. Eva Green (Certain intercalating dyes can be inhibitory to LAMP) for visual detection or if you have access to Real Time PCR instruments run amplification profile followed by specific melt point to check. In our Lab we currently employ commercially available OptiGene mastermixes in conjunction with GENIE II instrument. The OptiGene mastermix contains strand displacing enzyme Gsp which is far superior to Bst speed wise
Hi James,
Thank you for the suggestion. I have another question though. I am also using SYBR Green I for fluorescence detection (which I add to the tubes after amplification), and I read that adding this dye before amplification will hinder the amplification process itself. What do you use to detect your LAMP results?
Hi Maria
We have successfully used Eva green in the past. We have had problems with SYBR green 1 addition pre-amplification although other authors report no issues... I refer you to following paper which gives some excellent info on selection of fluorescent reporter dyes for Real Time LAMP (Seyrig et al. 2015 J. Micro Meth. 119:223-227. For simple end point visual detection I can also recommend Calcein dye commercially available as Florescent Detection Reagent (FDR) from Eiken. You can also make this up in house.
Hi James,
Great discussion.
I am facing some problem, whenever I set up LAMP reaction it is working with only few DNA concentration. The entire reaction mix is same except the DNA concentration. It worked with high and low concentration but not with intermediate concentration, first I thought may be some pipetting error but I observed the same for at-least 5-6 times. what is going on?
Thank you.
Hi Bhagya thats a tricky one! Has all your target DNA been prepared/extracted the same way?
your listed primer concentrations are about 25X too high, which could definitely contribute to non-specific amplification.
It may be occurring dimer formation, I recommend evaluating different concentrations of F3 / B3, starting from 0.2 micromolar to 0.5. I recommend making several combinations as: (F3 / B3) corresponding to (0.2 / 0.2); (0.2 / 0.3); (0.3 / 0.4) and so on. I hope have helped!
Hello,
An easy alternative that I have found useful, is replacing betaine for DMSO (check attached paper). For certain applications it has provided better results. Also I agree with Nara regarding the primers concentration optimisation.
Good luck.
http://www.mdpi.com/1420-3049/20/4/6048/htm
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