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16s rRNA giving weird results.
#1
Hello.

I've been having some recent problems while doing the 16s rRNA procedure. I am testing unknown samples of bacteria, which I will sequence later. I isolate the DNA directly from agar plates taking approximately 20 colonies using Qiagen DNeasy spin columns.

The problem is some of the samples are showing as negative on the elecrophoresis gel, while the negative control is positive. I have done it 3 times, and it always gives the same result. I am very careful and I doubt it is contamination, and if it is contamination it would be likely that the other samples would be falsely positive as well.

I am therefore wondering if it could be that the negative control has a smaller volume than the real samples. The samples are 15 ul each, but the negative has 12 ul since it has no template DNA. I enter the total volume as 15 ul on the PCR machine, and I am not sure if that can make a problem for the negative control.

I highly appreciate any answer to this!  Big Grin

Thank you.
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#2
Hello Amoeba,

I think before running gel electrophoresis, you should quantify DNA for each sample along with the negative control. Also, take blank buffer/distilled water as a negative control. For samples, showing negative for DNA, you can take more number of colonies, to ensure sufficient amount of DNA extraction.

--AD
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