Exact amplification in negative control

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Exact amplification in negative control

Postby astroboy89 » Mar 22 2018 6:23 am

I am working with transgenic plants and use genomic DNA PCR screening to search for positive transgenic plants. I use the plasmid containing construct used for transformation as positive control , wild type untransformed plant as the negative control and transformed transgenic plant as samples. I am facing a very annoying and frustrating problem. I am seeing the exact band I want in all my samples including the negative control where it should not be seen at all. I have isolated genomic dna of the wild type plant a different day than all my transgenic samples to avoid cross contamination.I always prepare master mix and when adding template I first add the control followed by the others. I have also used bleach to wipe the work area (laminar hood) and pipettes and then UV'd it for 5-10 mins.Then I start working. I have used fresh batch of buffer,dNTPs and Taq polymerase. I am using different combination primers such gene, gene+terminator, GUS gene etc. All give the same results, exact size band in the negative control. I am close to tearing my hair out and this has been going on for close to 2 months. I am at my wit's end and dont know what to do. I have also used comntrols such as without primer and without template. Amplification seen in without template control. But I have used fresh primer stock.

Please help me identify the source of contamination and eliminate it as soon as possible. What can I do ? :cry:
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Re: Exact amplification in negative control

Postby relaxin » Apr 06 2018 1:42 pm

Is the size of the amplified band corresponding to the one expected?
Do you use hot-start polymerase or use wax layer to separate the polymerase and the rest of reagent?

I suspect that what you are seeing is primer-dimer.
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Re: Exact amplification in negative control

Postby 29yrsExperience » Apr 06 2018 8:25 pm

A few more things to consider (forgive me if any seem too obvious to mention). Are you using factory-sterile aerosol-barrier pipette tips? Do you open your amplified tubes and run your gels far away from where you set up the reactions? Do you use a separate set of pipetters for post-amp handling? What kind of water are you using and have you changed water sources? I’d quit messing with my samples until I got the + and – controls working properly. Oh BTW, how dilute do you make your plasmid pos. control template? I’d cut it down to maybe 100 picograms/ul.

Did you get amplification even in a “no primer” control? That is weird. Try also a no amp control- a rxn you don’t even put in the PCR machine- and see if there is anything in there that shows up on a gel.

When you say “exact size band” does this mean that for each different primer pair, you get its specific expected band size? Are they different sizes? What sizes are we talking about here?

Hope we can help!
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