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Exact amplification in negative control???


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#1 astroboy89

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Posted 21 March 2018 - 10:26 PM

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: 


#2 bob1

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Posted 22 March 2018 - 06:01 AM

It sounds like a mass contamination of possibly multiple sources. This is most likely either from your plasmid positive control, or is from PCR product.

 

Your best bet is to get rid of all of your PCR stocks, get new everything and start afresh. You should strongly consider (if you can) shifting to a new room, and using a new (only used for mastermix) set of pipettes for setting up the mastermix, then coming back to your old room to add DNA. At a minimum, I would ensure that you have a set of pipettes that you only use for setting up the PCR, and another set that you only use for PCR products.

 

In my experience, laminar hoods, while useful, do not prevent PCR contamination, and in many cases can actually add to it! This happens because they blow air around - the filters are not capable of removing molecules from the air, so passing more air over your sample increases the chance of having some aerosolized DNA ending up in your reactions.






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