(LAMP) Problems with Loop-mediated isothermal amplification

[msarah]

HNB never affected our reactions at a concentration of 120uM final. We prepare a 20 mM solution in DDW and add 0.15 uL to a 25 uL reaction. HNB from Sigma cat # 219916 - 20 mM solution prepared by adding 0.01197 gm to 1 ml DDW.

[judsonzhao]

Hi all,
I've read all your post, but still not so clear how you can solve the FALSE-POSITIVE problem.

That is the main issue I concerned about.

I think my primers are good, have used them for years. All my positive come with positive, occasionally I will encounter with a false-positive. In gel, it just has a light band but not ladder-like pattern.
I have totally no idea why they come as false-positive. Because I once prepare all reagents in one tube, then distributed 3/4 of the mixture to 3 others which were negative controls, and only the one I used to prepare give me false-positive.
So I prepared them separately, but can still get one false-positive.

But sometimes, it just runs so good, positive comes to positive, negative comes to positive.

Any idea?... Really appreciated~ Hope to discuss with you guys, hopefully we can all get rid of these issues!

[jensf]

Hey, what is your melting profile? I always got a wide but small peak at 55°C to 70 °C and one bigger at 90 °C.

[deen23]

Hello all,

I'm trying to make a fluorescent detection reagent using calcein and MnCl2 (based on the Eiken LAMP Kit). It was hard to find out from papers how people have made their own, but I found two pieces of information:

1. The final concentration of calcein in a 25uL rxn mix should be 25uM, and of MnCl2 should be 0.5mM.
2. Calcein is dissolved in DMSO, and then mixed with MnCl2 dissolved in water.

I've tried (with both MnCl2 tetrahydrate and anhydrous), and unfortunately the final solution starts fluorescing immediately. I was wondering if anyone was successful in making this?

Thank you!

[Babak]

Has anyone used Picogreen for LAMP detection. Can you please provide Picogreen volume in 25 uL of solution?


Thanks
Babak

[JMCK]

Hi all
I have just discovered this forum I have some previous experience with LAMP assay development and the one thing I learned the hard way is never ever open the tube post amplifcation. No matter how careful you are you run a serious risk of contamination which will result in the inevitable false positives! Which are infuriating as just when you think you have it under control....The literature is replete with gel images, restriction digests addition of intercalating dyes after amplifcation etc. these should come with a serious health warning!
Anyways someone asked for a homebrew Calcein recipe here is one I have used successfully

Make a stock 2.5 mM solution of Calcein in Nuclease Free Water. Make a stock 50mM solution of MnCl2 in nuclease free water. To make 100ul of FDR (Calcein) mix 50ul of calcein and 50ul of MnCl2. Add 1 ul per LAMP reaction Final vol=25ul). I have successfully frozen the FDR and it works very well. Regards J

[Madray]

'm trying to make a fluorescent detection reagent using calcein and MnCl2 (based on the Eiken LAMP Kit).

[jensf]

Hey,

i had he same problem when solving calcein in DMSO. Try to solve most of calcein in water, or maybe try glycerol and then make your stock solution.

Does anyone get rid of the false-positive due to contamination by changing work flow?

Originally Posted by deen23

Hello all,

I'm trying to make a fluorescent detection reagent using calcein and MnCl2 (based on the Eiken LAMP Kit). It was hard to find out from papers how people have made their own, but I found two pieces of information:

1. The final concentration of calcein in a 25uL rxn mix should be 25uM, and of MnCl2 should be 0.5mM.
2. Calcein is dissolved in DMSO, and then mixed with MnCl2 dissolved in water.

I've tried (with both MnCl2 tetrahydrate and anhydrous), and unfortunately the final solution starts fluorescing immediately. I was wondering if anyone was successful in making this?

Thank you!

[treebell23]

There are an increasing number of people that will be choosing to deal with unwelcome solutions in an friendly to the environment fashion. It is important

[deen23]

Hi all!

My problem with making my fluorescent detection reagent (based on the Eiken kit) was due to a pH problem! It was too acidic before (a much lower pH than that of the Eiken kit FD). Instead of dissolving MnCl2 in water, I dissolved it in Tris-HCl (pH 8.0). The calcein was still dissolved in DMSO. After mixing the two, it turned a dark orange/brown colour (instead of bright green), and matched that of the Eiken kit FD. I used it in a LAMP reaction, and it worked. My negative controls remained orange, and my positive controls turned green.

Hope this helped.