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(LAMP) Problems with Loop-mediated isothermal amplification

(LAMP) Problems with Loop-mediated isothermal amplification - DNA Techniques

(LAMP) Problems with Loop-mediated isothermal amplification - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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  #11  
Old 07-20-2011, 10:35 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hi Nickdn
I do not understand how Q-LAMP, with real time fluorescence reading on FAM channel helped solve the problem. We found that the source of the contamination was one of the primers. At the moment we are working with single frozen mixes of most of the components with no apparent problem with contamination. Our new problem is that we do not seem to get consistent repeats from reaction to reaction. Have been titrating our DNA and ca see the decreased signal and then suddenly is goes up or disappears completely. Very hard to depend on this. Any ideas??
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Old 07-20-2011, 02:51 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Thank you for your response! I am looking at the OptiGene designer now. Do you need to buy it or is it free to download?
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  #13  
Old 07-21-2011, 12:57 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hi msarah,
We used normal LAMP before, that ment we had to open the reaction tubes after amplification. We have limited space, we used same workstation for reaction mix and sample analysis. We figured it had to be room contamination, because when we switched to new primer stock and reaction mix we still had false positive results. I never titrated DNA but amplicons all looked very similar after gel Gel electrophoresis analysis.
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Old 08-17-2011, 01:53 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hey,

so it seems to be a general problem. Everything indicates that it is kind of contamination. During LAMP huge amount of DNA is amplified and also the contamination.

The result of my experiments are all positive. On Gel, you can see typical ladder-like structure, for positive and negative.
The same in Real-time LAMP with FAM channel. Positive and negative getting positive after 25 to 35 minutes, the negative few minutes later. Melting curve analysis with sybr green indicates a wide peak between 55 and 70 C and one peak at 89C. The smallest product in LAMP is the dumbell-structure (starting point for exponential amplification) The calculation of melting temperature of this target region also showed 85 to 89 C. The LAMP seems to work well but there is some contamination.

I changed LAMP reaction mix.

So it might be possible that the contamination is in BST-Pol or in Primers. But PCR results are okay. I have no idea yet.

I also tested the LAMP without one of the inner primers. When one or two of the inner primers are missing, LAMP is still working. When inner Primers are missing, LAMP doesn't work. This is logical because inner primers are forming dumbell-structure.

Does everyone solved the problem so far?

best regards

JF
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  #15  
Old 08-18-2011, 08:12 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

I am just as confused as u. When we were having problems with "false positive" we remade all our buffers and ordered new (same sequence) primers but kept the Bst-pol. We use very strict cleaning protocols and have divided the buffers into small aliquots before freezing. The problem seems to have solved itself (hopefully). What worries me more is that the ease in which the system gives false + seems to indicate that the method is not as highly specific as we have read. When you say inner primers are you referring to FIP/BIP or the loop primers.
The problem that we are battling now is a method to review the results w/o running a gel. We do not get enough sediment for a easy analysis. HNB color change is not consistent, calcein is quite weak. I read in an article in which CuSO4 is added to the reaction to increase the amount of sediment. Did not work in our hands. Any input?
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Old 08-24-2011, 12:42 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hi
I've just started my LAMP and I just keep getting false-positive reactions with the calcein even though The reaction doesn't have primers or DNA. What can I do?
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Old 08-24-2011, 12:47 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

I've started my LAMP experiments. I'm using calcein an is giving me false positive even though there is no primers or DNA in the reaction. Has anyone experience the same??
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  #18  
Old 08-28-2011, 10:19 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Have you solved your problem yet. We are running two different primer sets for completely different target genes. One consistently gives us good results and the other has false positive. The Bst pol is the same for both runs. I am beginning to think that the primers might be reacting with each other in some way. We use the lower concentration of Betaine 0.8 M which increases the stringency. The next step in our plan is the move the primers slightly and check very carefully that there is absolutely no change that they are annealing to each other and giving the false positive. As far as Acinaria having + results with no primer present is very strange. We had positive results when no DNA template was present , but - results when BST or dNTPs were not present. this leads me to believe that the primers are reacting with each other and have to be replanned.
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  #19  
Old 08-29-2011, 07:36 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

to check if your primer are interacting with each other you can make a control with just water instead of DNA and run in a gel. If u can see band that will be ur primers.
I'm running different Mg and Mn concentrations for my buffer apparently calcein is reacting with my Mg concentration... still need to do more test :s
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  #20  
Old 08-29-2011, 11:07 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Their are two possible reasons for false+. Contamination in reaction or primers are not working well.

The detection with calcein / pyrophosphate sedimentation after centrifugation or agarose-gel-run shows similar results for + and - sample! The ladder-like structure can be seen for both. After 25 to 35 Min. you can see the colour change from orange to green. This is similar to results found in papers (real-time LAMP with turbidity).

Is it possible that bad primers (primer annealing) form similar ladder-like structures during LAMP without DNA as target?

Who has some experience with LAMP?
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amplificaion , amplification , dna , isothermal , lamp , loopmediated , primer design , problems


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