I'm trying to use calcein as a detection dye for my LAMP assay, but am having trouble. I've tried titrating it out against MnCl2 and tried adding it before and after amplification but have only achieved successful colour change once. The colours are either not changing between positive and negative (all are orange) or the colour change is opposite (positives appear orange, negatives appear green). I have ruled out contamination of the assay as a cause. Thanks.
Hi
I have used the following in house Calcein dye preparation successfully for a number of LAMP assays :To produce 100µl pre-made LAMP Calcein Fluorescent Detection Reagent (FDR) ; Mix 50µl of Calcein (2.5 mM) + 50µl of MnCl2 (50mM). Then add 1µl of FDR to each LAMP reaction (Final volume 25µl). Provided your LAMP assay is working (Are you seeing visual turbidity in reaction tubes?) this approach should work well. Regards James
I think, James suggestion should help you but check if you have ladder-like product for positive controls and lack of it for negative on regular agarose gel stained with EtBr or SybrGreen or equivalent dsDNA staining dyes. The best if yo do agarose gel in lab far away from lab where you set up your LAMP reaction. The LAMP yield is higher than PCR and product contamination can be a really serious problem.
Krzysztof gives good advice but as he suggests please be careful when carrying out any post amplification manipulations of LAMP products! Good Luck
Thanks James, That's really good advice and I will give it a go. The LAMP assay itself is working, but detection is an issue. And thanks to Kryzstof and Ahmed for the contamination advice, I know all too well how susceptible LAMP is to it! Thanks everyone!
What it is that you should have all your primers (about 4 to 6 of them) in good working condition. Also try to optimize your reaction temperature, chose between 62 -64 degrees. For your negative control, I suggest that you use ultra pure water as template instead of using a known negative DNA sample alone. After your LAMP reaction I suggest that you try James formulation. However you can also resolve your product on agarose gel stained with gel red which work perfectly for me or you can decide to change your detection dye completely and use 1 µl of 1/10 dilution of SYBR Green I (Invitrogen) after your reaction for the detection of positive amplicons. You should know that if the reaction does not work well detection will be a problem as it is common feature of LAMP reactions.
Hi,
Did anyone try using hydroxynaphthol blue? I was trying for that, but couldn't get a proper result.
One important thing concerning calcein. Concentrated stock (between 1-5 mM) of this reagent should be prepared in DMSO, since calcein is poorly soluble in water. Dissolving in alkaline environment (for example in 1M NaOH) is possible up to 50 mg/ml but this is beneficial if you want to titrate calcium, not in LAMP reaction!
That is good advice from Krzysztof although it can be achieved Calcein is tricky to dissolve in water. Please, please try and avoid use of gels if at all possible this causes the most issues with contamination!
about one year at -20. You can also make ready mix of calcein and MnCl2 as James recommended before and store that FDR in aliquots at -20.
hi Krysztof .....i also want to give some suggestions if it is helpful.....while making use the concentration of calcein mncl2 like this 50 mM Calcein-500 mM MnCl2.
while making calcein, add 1M NaOH to dissolve, and put in shaker overnight but then remember to adjust the pH of calcein 7-8. then you can make the desired ratio of calcein mncl2(1:5, 1:8, 1:10).....i am attaching a publication in the attachments, i hope so it will be helpful.
Dear Leanna Sure
In high concentration of Mg/Mn (16:1) ratio, resulted in high background fluorescence in negative control. You could change this ratio to 4:1 according to previous studies or change your stain to Gel Red 1/10000 x or another stain.
I suggest you that read this article for solve problem. You success with decrease ratio to Mg/Mn 4:1.
Can someone comment on the dye used by NEB for colorimetric LAMP?
One of my team members has tried NEB for colorimetric LAMP recently, but in our case, this kit did not work for untreated sap from virus-infected potato plants. It worked for sap boiled for few minutes (color change occurred for virus-infected sap vs sap from the healthy plant) but sensitivity was very low compared to other company kit. We will check if this kit needs some optimization of the sample buffer to boost sensitivity.
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