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Amplification in negative control

Amplification in negative control - PCR - Polymerase Chain Reaction Forum

Amplification in negative control - 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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Old 05-21-2011, 01:31 PM
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Unhappy Amplification in negative control



Hello friends, plz help me. I'm getting bands in negative control and that too of the exact expected size as in positive. I have tried everything from changing components till changing the set of primers (I orderd a new in fact). still I am getting bands in no DNA controls, WHAT TO DO?
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Old 05-21-2011, 05:56 PM
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Default Re: Amplification in negative control

Try to prepared the negative control before you even handle any sample DNA, seal the tube after that and do not open it until gel electrophoresis (or whatever you do after the thermal cycling is over).

ALWAYS clean the PCR work place with 70% etoH or diluted bleach and try to avoid air currents while handling opened reagent or sample tubes. A hood with UV light is useful: You can irradiate the surface for 20 min before working. You can even irradiate some of the PCR reagents such as water, PCR buffer and MgCl2 (other reagents are UV sensitive), as well as any equipment you use like pipettes, tip boxes and such.

Another tip is to try not to touch the surfaces of the tubes with the pipettes (just with the pipette tip) as this is a potencial cause of reagent contamination.

Hope this helps.
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Old 09-07-2011, 06:09 PM
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Default Re: Amplification in negative control

I have a similar problem from time to time. Two techniques that seem to help are using proper pipetting to avoid blow back of any contamination and keeping the reaction cold during preparation. I have ruled out all other sources of contamination.

I am comparing two different strains of insect DNA. My standards always work fine and the negative control is always one and not the other strain. If the problem were simply contamination, I would expect to have the signal sometimes appear to be one strain and sometimes the other strain. I'm now wondering if my primers need to be more diluted, although that is more likely the solution to primer dimer problem, which is not what I am experiencing.
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