We did a genomic extraction (Qiagen) on
Stachybotrys chartarum and tried to measure the concentration in my eluate using a NanoDrop. I don't believe this is giving me an accurate reading whatsoever and on top of that there seems to be a good amount of EtOH contamination based on the 230 reading.
We then conducted PCR using a set of primers and PCR protocol described in the literature to amplify a segment of DNA that should be ~136bp long. We then ran this product on a 2.5% agarose gel. (attached images)
I have tried this multiple times varying the amount of template DNA used (undiluted, 1:10 dilution, 1:1000, 1:10000, 1;100,000) and each time there is a lot of DNA stuck up at the top of my gel/in the well, a large smear (can see many individual bands if the image is adjusted), and no desired product at 136bp. This doesn't make sense to me as the 1:100,000 dilution of template should have considerably less DNA and I can't imaging that my primers are nonspecifically binding in such a way that the entire genomic DNA is amplified.
What could be causing this and how can I avoid this and ensure I get my product? Thanks!
Dear Parisa, only genomic DNA is seen in your gel. Firstly, you should check your primers and use a positive control DNA. After that in serial DNA concentration, you can optimize template.
Best regards.
Dear Perisa,
I recommend you to use the Boiling Prep method, it's very easy (Article Specific detection of Stachybotrys chartarum in pure culture...
). So, as Mert says, you should evaluate your set of primers and use a positive control.Best regards.
Hello Parisa,
Adding to the suggestions given already, try adding only 50 or 100ng of DNA template per PCR reaction. Also for a product of around 150bp you can use 1.5% Agarose gel.
If you feel your sample is contaminated with EtOH, try concentrating your whole sample (using a concentrator). This will remove all the unwanted EtOH. Let us know if it works.
Best!
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