(LAMP) Problems with Loop-mediated isothermal amplification

[telemartnetwork]

Thanks for starting this topic. I received good info & useful as well from here. It is good to be part of this community.

[Lampboy]

Bst DNA Polymerase at 8U for the rxn, or 0.32U/uL

BST Has NO RT activity, you have to use a seperate RT enzyme, problem seems to be that the amount needed vary's upon each assay, sorry you have to do a titration and find out what level suits that assay, 0.2 Units upto 2.5 units RT enzyme per reaction.
AMV Reverse Transcriptase from Invitrogen works well in my hands, I saw an ad for optigene was quoting their kit has RT activity, yet to try it tho!

[jensf]

Hey,

in my case i found that the combination of reaction temperature with the primers was not optimal. Here I had to increase the temperature above the "optimal" of Bst-Pol. At the moment my primers are working and NTC is correct. But now my Calcein-Detection is not working...i use less primers and dNTPs as standard LAMP. To distinguish POS and NTC i use SybrGreen.
The False-POS in my case were due to non-specific amplification of inner primers (FIP/BIP)

[eproulx]

Hi, new person here.

I've just started working with an LA-500 LAMP device, and I'm the first person to use it in my lab. I'm a first year co-op student, so naturally the first assumption we're all making is that I've been sloppy with changing pipette tips and such, but as time passes that's looking less like a possibility.

We ran into some early issues where all results were coming out negative. This turned out to be an issue with insufficient vortexing of the buffer (we're using pre-mixed Thermopol Reaction Buffer from Lucigen), resulting in poor function of the Bst Pol. So, we fixed that problem.

Then I started getting false positives. After about a week and a half, we put aside the primers we had been using (for eae and stx2 genes) and started fresh with stx1 primers and all new reagents. At the same time, I began preparing master mix in a BSC, and aliquoting it and template into reaction tubes in a separate laminar flow hood used on for that purpose.

That was working great for about 3-4 days, then random false positives started popping up again. At this point we're back to full on contamination.

JMCK and jensf's advice makes a lot of sense to me. In the confusion, I've been checking my LAMP product on gels, and then putting those tubes back in the same -20 freezer as my reagents. Since I'm being so careful to prepare everything in hoods, douse everything in DNA-Away and make sure I'm not just mixing template into my sterile water, contamination of the reagents in this manner seems like one of the only options.

Aside from that, when it works, it works well.

Any other ideas about what could be wrong here?

[biogal]

Originally Posted by jensf

Hey,

in my case i found that the combination of reaction temperature with the primers was not optimal. Here I had to increase the temperature above the "optimal" of Bst-Pol. At the moment my primers are working and NTC is correct. But now my Calcein-Detection is not working...i use less primers and dNTPs as standard LAMP. To distinguish POS and NTC i use SybrGreen.
The False-POS in my case were due to non-specific amplification of inner primers (FIP/BIP)

What temp and concentrations are you working at

[zizu]

hello! i'm a PHd student trying to set up a lamp protocol for T. gondii...one very basic question what's the difference between the Bst polymerase full lenght and bst polymerase large fragment?
which one is to be used in LAMP? thanks everyone!!!!!!!!!!!!!!!!!!!

[jlamp]

Hey i use normal concentrations, given in all papers...it's very hard to get rid of cross-over contamination and false-positive..I think this is most serious problem in LAMP. Someone has new results?

[MCaldera]

Hello there!! I also working with LAMP with a bacteria in rice called B.glumae .We designed the primers with primer explorer v4 and Im using MnCl2 and calcein for fluorescent visualization.The problem is that my negative control(water with the reaction mixture) produce fluoresence.Do u have any good idea whats going on ?

thank u so much

[jlamp]

Originally Posted by MCaldera

Hello there!! I also working with LAMP with a bacteria in rice called B.glumae .We designed the primers with primer explorer v4 and Im using MnCl2 and calcein for fluorescent visualization.The problem is that my negative control(water with the reaction mixture) produce fluoresence.Do u have any good idea whats going on ?

thank u so much

Hey, most time its due to cross-over contamination from last reaction, as decribed previously here...

[sittingchair]

Hello!

Also performing routine LAMP. However, I am using my own buffer (Tris-HCl pH8.8) and HNB for colorimetric visualization. The problem I am encountering now is that when I add the HNB to the master mix, it immediately turns to a sky blue (positive indicator). I have tried various concentrations (variations from .003mM to 120mM) but they all produce the same result.

Thanks a lot for any suggestions!