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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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  #91  
Old 04-20-2013, 10:05 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Hey friends,
I have an odd problem. I dont know what has happend not only I dont get false positive but also I just get false negative. No reaction occure in tubes. Does any body have any idea?! Every thing was OK at th first but now with the same reagents I dont get any LAMP reaction. I can not figure it out what is wrong with it.
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  #92  
Old 04-20-2013, 10:07 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

If the PROBLEM IS with MY ENZYME WHAT HAS HAPPEND TO IT?????!!!

Any reply is valuable.
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  #93  
Old 04-20-2013, 04:02 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Heyyyy any body here!!!!!
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  #94  
Old 04-22-2013, 01:32 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

If the problem is the enzyme, It could be because it degraded, most probably because it was left out of the freezer (I mean quite above -20C) for an extended time. UV light exposure can also render the enzyme useless.

If there is anyone else working in your lab ask them to use your enzyme in one of their reaction (few tubes: 3,4) to check if that is the problem.

I hope this helps you nikan.

Good luck
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  #95  
Old 05-14-2013, 09:37 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Dear friends,

I found that the PCR is more sensitive than my LAMP assay. Does anybody encounter similar problem? How to increase the sensitivity of the LAMP assay? Thank you.
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  #96  
Old 05-14-2013, 03:26 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Quote:
Originally Posted by nikan View Post
Hey friends,
I have an odd problem. I dont know what has happend not only I dont get false positive but also I just get false negative. No reaction occure in tubes. Does any body have any idea?! Every thing was OK at th first but now with the same reagents I dont get any LAMP reaction. I can not figure it out what is wrong with it.
Use PCR to check the quality of your DNA samples. If there is positive result with PCR, then there might be some issue with LAMP reagents. U can replace the MgSo4 with Mgcl2. It works in my assay.
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  #97  
Old 05-15-2013, 05:57 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Quote:
Originally Posted by aahu View Post
Dear friends,

I found that the PCR is more sensitive than my LAMP assay. Does anybody encounter similar problem? How to increase the sensitivity of the LAMP assay? Thank you.

Hi aahu,
I can come up with some reasons. The first is your enzyme, it's efficacy may have been decreased beacuase of any reasons including bad storage condition. Secondely, Your primers may be dagraded not totally since you have your reactions performed. Some times this happens in molecular process especially in PCR, at first you see sharp bands,then with the same condition the band in your gel gets pale and pale till it disapears. dNTP mix can also be affected by so many freez thawing. decreasing strigency may help for example by increasing MgSO4. Any how to tackle you problem I suggest you work with new reagents if possible.

Good luck

Last edited by nikan; 05-15-2013 at 05:59 PM.
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  #98  
Old 06-04-2013, 07:36 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

hey sarah how do you prepare your calcein and your HNB ?
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  #99  
Old 06-04-2013, 07:38 PM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Does someone has used Ethidium bromide for LAMP fluoresence?
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  #100  
Old 06-06-2013, 05:44 AM
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Default Re: (LAMP) Problems with Loop-mediated isothermal amplification

Quote:
Originally Posted by MCaldera View Post
Does someone has used Ethidium bromide for LAMP fluoresence?
Hi MCaldera,

I have used EtBr for my LAMP assay. Positive sample will turned into pink whereas negative will remain orange/red.

Load 10 ul of 1% (or less) Etbr into your LAMP product. Good Luck
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amplificaion , amplification , dna , isothermal , lamp , loopmediated , primer design , problems


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