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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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  #111  
Old 06-16-2014, 03:21 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

I have some problem with LAMP reaction.
I've carried out LAMP reaction with HNB and Calcein/ MnCl2 but they were unexpected results.

With HNB, when I did not add HNB to the reaction, it occur certainly with serial bands in Agarose gel. But when adding HNB, the reactions had not happened. the component of both reaction is the same. Did HNB inhibit the reaction ??

With calcein/MnCl2, nothing were observed.

Can you help me to explain ?
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  #112  
Old 07-02-2014, 06:48 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hello!

HNB at a final concentration of 120 uM is consistently reported to have no inhibiting effect on the LAMP assay. You should try to repeat the assay with this concentration doing two variations of a MgSO4 gradient: one with HNB at 120 uM and one without. Prepare the master mix for both experiments together, then divide it out.

I'm also working on a LAMP assay and here is my problem: no ladder banding has been observed so far, however I am picking up a single band at around 4.5k in all of my positives.

I blasted all combinations of primers on NCBI and found that a potential product of the F1c and B1c ends of FIP and BIP respectively could produce a product of 4.5k in length.

Any ideas of what could be going on? Is it possible that my primer ratio (1:4 outer:inner) is simply not optimized?

Thanks
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  #113  
Old 07-03-2014, 06:09 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Thank you.

Again, what the different between calcein and calcein AM? Could LAMP use both?

Is there any protocol for calcein and HNB preparation?
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  #114  
Old 07-03-2014, 02:16 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Quote:
Originally Posted by sittingchair View Post
Hello!

HNB at a final concentration of 120 uM is consistently reported to have no inhibiting effect on the LAMP assay. You should try to repeat the assay with this concentration doing two variations of a MgSO4 gradient: one with HNB at 120 uM and one without. Prepare the master mix for both experiments together, then divide it out.

I'm also working on a LAMP assay and here is my problem: no ladder banding has been observed so far, however I am picking up a single band at around 4.5k in all of my positives.

I blasted all combinations of primers on NCBI and found that a potential product of the F1c and B1c ends of FIP and BIP respectively could produce a product of 4.5k in length.

Any ideas of what could be going on? Is it possible that my primer ratio (1:4 outer:inner) is simply not optimized?

Thanks
@ Sitting chair:
I found something similar in my reactions (~4.5 kb band instead of ladder product) , I diluted gDNA template (only, w/o lamp reagents) as it was used in reaction (3 in 22l) and loaded it onto gel- in my case the band seems so come from template, however.
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  #115  
Old 07-03-2014, 02:25 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Quote:
Originally Posted by sittingchair View Post
Hello!

HNB at a final concentration of 120 uM is consistently reported to have no inhibiting effect on the LAMP assay. You should try to repeat the assay with this concentration doing two variations of a MgSO4 gradient: one with HNB at 120 uM and one without. Prepare the master mix for both experiments together, then divide it out.

I'm also working on a LAMP assay and here is my problem: no ladder banding has been observed so far, however I am picking up a single band at around 4.5k in all of my positives.

I blasted all combinations of primers on NCBI and found that a potential product of the F1c and B1c ends of FIP and BIP respectively could produce a product of 4.5k in length.

Any ideas of what could be going on? Is it possible that my primer ratio (1:4 outer:inner) is simply not optimized?

Thanks
For me it workes most of the cases when I melt template and primers together and THEN add LAMP reagents, which is of course not favourable, but you can try at least 90C for 5min, cool and add reagents
I am using the same Outer: Inner Primer ratio of 1:4.
Sometimes I get great ladder pattern, repeated another day it's just DNA smear on the gel
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  #116  
Old 07-04-2014, 03:59 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hello!

I am a student doing a thesis on LAMP assay for detecting MTB. I am curre tly having problems. I am having a lot of false positive results, like yesterday I ran 10 samples and all them were positive but my negative control was negative. Same is true for my previous lamp assays. Almost half were positive but my negative control always remains negative. Is it a contamination? but i pipette my negative control last. If it was a contamination wouldnt the negative control also be positive?
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  #117  
Old 07-05-2014, 06:10 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

1. Contamination should always be ruled out. Clean everything with 10% household bleach (3,5%) and try again.
2. If your negative control is molecular grade water try using DNA that is similar to your target DNA put does not contain the amplicon which you are looking for. See if this negative control turns positive...... this means that you are miss priming.
3. Poor primer design which results in primer self amplification is the most possible reason for false positives. Check and recheck the interaction of the primers with each other. You can do this by RealTime with sybrgreen.

Good Luck
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  #118  
Old 08-05-2014, 11:00 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

I wonder the mechanism of the HNB change from violet to light blue in LAMP reaction. Anyone can help me explain that? Thanks a lot !!
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  #119  
Old 08-28-2014, 07:14 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hello! I've been using LAMP for a while. I couldn't observe any turbidity with 8mM MgCl2 and all the usual reagents and protocol, however the bands could be seen on 2% agarose gel. However, the bands are somehow blurry and smeared with no distinction. Post-amplification staining with EtBr sometimes result in bright fluorescence, which is strange, since all products could be visualized on the gel.

What should I do to enhance turbidity?
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  #120  
Old 09-04-2014, 02:14 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

I have tried the Tomita et al concentrations of Calcein and MnCl2, the Goto et al concentrations in many attempts to try and prepare home made Eiken Detection Reagent (because i ran out and it takes forever to be shipped). Is there anyone who know exactly what goes in the Eiken Detection Reagent?

The pH of home made attempts can range from 6-8 (in order not to fluoresce) but still i find there must be some buffering agent missing because i get very spontaneous and sudden signal from samples where I wouldnt expect to see signal (together with PC).. Also I get irreproducibe results and observe an overall stochasticity.

So far I have tried dissolving Calcein in DMSO and MnCl in H2O or Tris-HCl pH7.5-8 and then supplement with H2O or DMSO or different combinations of both (1/4 H2O 3/4 DMSO, half half etc..)
I am not happy with anything and believe I m missing something Eiken Detection Reagent has in it...

Please help!!!




Quote:
Originally Posted by deen23 View Post
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!
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amplificaion , amplification , dna , isothermal , lamp , loopmediated , primer design , problems


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