Hi,

Check the pipette, clean it very will with ethanol 70%, then let it dry. another possibility is the humidity in the lab some you have a aerosol particles, work in a hood. good luck.

Hi

HNB color is pH dependent, check the pH of your reagents and buffer. If a single reagent is troublesome, you will see color change even if LAMP reaction has taken place.

Read the following article for details.

Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Motoki Goto1, Eiichi Honda2, Atsuo Ogura1, Akio Nomoto3, and Ken-Ichi Hanaki, D.V.M.

BioTechniques 46:167-172 (March 2009) doi 10.2144/000113072

a correction

read

even if LAMP reaction has not taken place.

Hi Mkhululi!

Im having the same trouble as you! were you also working with HNB? did you solve your problem!? anny suggestions ?

@Ruchi Singh the problem was not HNB changing colour when there was no reaction but getting amplification in negative controls with all the other reagents except template DNA. The amplification of coarse was detected by colour change of the HNB and also on gels typical LAMP assay amplicons were observed. We ran the gels after we observed the colour changes to check if the colour changes were due to pH and anything else besides DNA amplification, because we observed the typical LAMP banding patterns we deduced that the colour changes were caused by non specific amplification not changes in pH.

@Andrea Paz Donoso Youlton we then went on to find out what the source of non-specific amplification could be. We checked for contamination in all our reagents and working environment and we could not pick up any. Our main problem was that we were getting the same intensity in terms of both colour change in reactions with template DNA and also in negative controls. We got to a point where we were not sure if the amplification we were getting was of the template DNA or just our primers self amplifying. Because we could not detect any possible contamination we then thought the primer sets  we were using were self amplifying for some reason we could not understand. We did primer pair compatibility again. Some of our primers formed dimers with 3bps or more at the 3' end leaving a 5' overhang on the other primer. We thought this could be the problem. However, we had no means of proving that this indeed was the problem. We then went back to design new primer set and did primer pair compatibility for each set and chose primer sets that could not form dimers with 3' overhangs. We are currently working with the new primer pairs. I will keep you posted if we manage to resolve this completely. Please let me know as well if you find a solution.

 Mkhululi thanks for your answer! did you try ordering new sets of primers?  (the same primers you were using at first) Probably if it is a dimer or self amplification problem i wont see the usual bands when i see my electrophoresis gel, in my case I've seen the typical LAMP band pattern in my gel so i thought maybe one of my original primer  stocks got contaminated at some point. I've opened new buffer, MgSO4, dNTPs stocks ..etc. and still having the same problem so now i just ordered new primers and a new Bst. 

Also, sometimes it has happened that when i add HNB to the mix (the same stock and concentration as always) ... it just turns sky blue.. before any reaction!! Do you know what can be the problem there?? :/

Best regards!

Andrea 

Adrea it seems we have a similar problem. Yes, we ordered new sets and even changed suppliers with the original set of primers. We had primers targeting different genes and they all gave the typical LAMP bands on gels. When we analysed all the sets for primer compatibility they all seemed to share that common feature of dimers with 3' overhangs. Unfortunately we were not able to do melting curve analysis to check if indeed it was self amplification. I think melting curve analysis would be the best way to differentiate between primer self amplification and amplification of the target region.

Like I said before for us we will try the set of primers without  dimers with 3' overhangs and see if we can solve the problem that way.

Kind regards

Hi guys, I am having the same problem...

I did test my primers and reaction on a real time and my negative was okey. Now I am testing to get a colorimetric detection with HNB and the color get funny, I mean blue or violet  but when I run the gel I do have amplification in all the samples negative too!!! :(

I did realize tho, Andrea,  that if you use a concentration less than 6 mM of MgSO4 and add the HNB your mix stay blue!!

Nothing more at the moment that I can think about it to solve the problem...:(

Hi guys! Finally in my case i think it was a pair of primers that got contaminated so i just ordered new ones and now it has been working perfectly :). Rosa! yes I have been using higher concentrations of MgSO4 at it gets more violet .. also these days i have been using MgCl instead of MgSO4 and i think the colours look much better or easier to differentiate. Hey.. just realize you are in cambridge, I will be there monday and tuesday next week... any chance to meet you or visit your lab?? 

Best regards 

Andrea

I think my problem is in the buffer, but still I can't have a color detection, they all look violet! how much HNB do you guys use? 

thanks

I send you a prv message Andrea 

Cheers

Hi guys, I am also trying to use HNB to detect LAMP results. Though my experience is different. My HNB stays violet regardless the fact that I gel electrophoresis confirms positive result. I tried different concentration, and it is so that with 4mM or smaller amount of MgSO4 my HNB stays sky blue. I have learnt that HNB turns color as the Mg ion in the buffer is precipitated with the pyrophosphate ion released by dNTPs while DNA amplification. Yet I have no clue why my HNB is not changing color.. Can anyone give me some suggestions? Thanks a lot!

Hi Xiao,

I have the same problem, my HNB doesn't work properly. Can I ask you which kind of mix are you using?

I am using the otigene mix and with that one it is blue way before the reaction and stay the same color in all the samples ( negative included). while if I made my own mix using the Bst using different concentration of MgSO4 and DNTP I have "funny"color results..

will be nice to have a discussion

Hi Rosa,

Thank you for the help!

My reagents are from NEB (except for dNTPs from GH Health (illustra ))

My Buffer system are:

Tris-HCL ph 8.8 20mM

(NH4)2SO4 10mM

KCL 50mM

MgSO4 8mM

Tween 20 0.1%

dNTPs 1.4mM

Primers 1.6uM FIP/BIP 0.2uM F3/B3 0.4uM LoopF/B

Bst 0.32U/uL

WS RTx 0.3U/uL (I use RNA template from Qiaamp kit)

Also, there are 1.5uL enzyme buffer in the 25uL system:

Tris-HCL ph7.5 10mM

KCL 50mM

DTT 1mM

EDTA 0.1mM

Glycerol 50%

Triton X-100 0.1%

For your case, I think you should check the Mg ion concentration in your buffer, if the HNB stays blue even before reaction, chances are the Mg concentration in the buffer is lower than 4mM. You can try to increase it or just add HNB to a various of Mg concentration to see its color change.

Hi xiao, 

i thought so but you don't really know what is inside the mastermix cause is a company secret.. i think they have MgCl2 instead of MgSO4 or something else that change the pH, that's why it stay blue..

Anyway i also use the single reagents in a mix as you did the, Bst III and buffer from NEB and I am experiencing the same problem ( no color detection). I tried changing the concentration of  DNTP from 1-1.4mM and the MgSO4 from 5 to 8mM and  HNB from 150 to 200uM but nothing.... beside a problem of contamination in the Negative, that doesn't occur when I use the optigene mastermix adding only water, betaine and my primers.

How much HNB are you using and which brand ( i am using the Sigma one)? I am starting to think could be this or the Bst from NEB.. I am looking for other company that sell the same reagents to double check my mastermix.