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Nested PCR Contamination - URGENT

Nested PCR Contamination - URGENT - PCR - Polymerase Chain Reaction Forum

Nested PCR Contamination - URGENT - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 01-06-2009, 02:27 PM
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Angry Nested PCR Contamination - URGENT



hi!

i'm doing nested pcr. the first time i did it i had contamination in the negative control. i changed the taq (Hotstar), dNTPs, water and order new primers. i also cleaned the pipets, changed the tubes and the tpis (with filter).

it worked for a while but now i'm having the same problem. i always put the material in UV first. i have changed again the dNTP, water and made the primers from a new aliquote stock. the only thin i didn't changed was the taq (i did't have a new one). the problem continues!!! i made a 3 negative controls and only 2 were contaminated.

i'm getting frustated...

if you have any idea of what i can do to get rid of contamination please tell me. Thanks
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  #2  
Old 01-20-2009, 08:56 AM
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Default Re: Nested PCR Contamination - URGENT

Hi Nested welcome to the site,
Have you checked your pipettes? They do suck up micro droplets of DNA from mini-prep, midi-prep, bacterial stocks etc. and all it takes is someone to spit it back out.

Try to use filter tip pipette tips, and/or try different pipettes that are cleaned well and just used for your labs PCRs.

let us know if you find the source of the contamination will give me ideas if i ever get it again
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Old 02-22-2009, 02:41 PM
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Default Re: Nested PCR Contamination - URGENT

hello, i also have problems with nested pcr. my negative controls are always with contaminatio. Actually i don't known what to do, because i tried to change many things: i change gloves after every sample, i have pipetmans and pipets, which i use just for this stage i clean everything, use uv, but negative control the same, whith contamination.
Can you help me what to do, how to solve this problem
thank you very much
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Old 02-25-2009, 08:51 AM
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Default Re: Nested PCR Contamination - URGENT

Perhaps the problem was in extraction step?
I had the same problem and it was a contamination in extraction step.
What is your 1rst stage? Is a PCR or RT-PCR (one step)?
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Old 03-12-2009, 04:46 AM
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Default Re: Nested PCR Contamination - URGENT

Your contamination meaned that the negative control could produce the same PCR band as the samples?
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Old 03-13-2009, 04:20 PM
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Default Re: Nested PCR Contamination - URGENT

Your problem sounds similar to another poster, to which I've posted a reply:

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