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Problems with Loop mediated isothermal amplification

Problems with Loop mediated isothermal amplification - DNA Techniques

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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  #1  
Old 05-21-2011, 04:52 PM
Pipette Filler
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Default Problems with Loop mediated isothermal amplification



Hi everyone. I am a student working currently working on a master project with Loop mediated isothermal amplification (LAMP). I am really really desperate now as the water negative control and positive control have random patterns of positive and negative signals. Negative water control frequently turned green which is a positive sign after the addition of SYBR Green I. I have excluded the possibility of contamination but would it be that the primers are so nonspecific that they bind to each other and amplified by the Bst polymerase? I have tried reducing dNTP concentration and MgSO4 concentration as well as adjusting Bst polymerase conentration but it doesnt seem to make a difference. I ran out of money for redesigning primers (with the fact that there are not much options provided by the jpexplorer4). What else should I try for my optimization or did I missed out anything that could possibly lead to such a frequently false positive water control? Appropriate if anyone can reply. Thanx so much.
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Old 05-25-2011, 02:19 PM
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Default Re: Problems with Loop mediated isothermal amplification

I am also working on a LAMP assay for my project.

Originally, I was getting no amplification. Instead of the recommended 8U of Bst polymerase I added 16U. This gave me amplification with a clean negative Sigma water control. However, I could not repeat this result. Like you my negative control keeps coming out positive!

Have you been using Betaine in your reaction? If so, Have you tried changing the concentration?
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Old 02-01-2012, 11:43 AM
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Default Re: Problems with Loop mediated isothermal amplification

Hello Nocole and mcalapai, as I can read your threads, you had a similar problem as I do. Negative control turns positive with the addition of an intercalating dye. Meanwhile I am not able to detect the bands on the gel with the set of primers I am using. How did you solve your problems? I am getting desperate! thanks!
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Old 02-08-2012, 01:21 PM
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Default Re: Problems with Loop mediated isothermal amplification

I haven't yet! Actually there is a thread on here that is more active and has a lot of suggestions

If you look up loop mediated isothermal amplification problems it will pop up if you use google. It won't let me post the link on here.


Read through and see if any suggestions on there helps. I know though it is important to optimize your betaine and MgSO4 concentratrion for sure. Good Luck!
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Old 03-07-2014, 04:56 PM
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Default Re: Problems with Loop mediated isothermal amplification

agreed..
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