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cDNA degradation within a week at -20?

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#1 Sakumi



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Posted 12 June 2015 - 12:25 PM


I am in a bit of a pickle, again.

So I have been running RT-PCR (old fashion end-point, agarose gel) for a while now and accumulating data. To prepare for a presentation next week, I want to make a "representative data" figure of the bands. I have saved the cDNA and PCR products that I made on June 5th and that gave me good results.

First, I tried running the PCR products again on the gel, in the conformation that is needed for the figure. I see very faint bands, so I think perhaps they have been contaminated and degraded?

Then, I re-did PCR using the frozen cDNA and ran them on a gel. I see one cDNA sample that gave me a solid band, but everything else was very faint.

So while I don't remember if I have freeze-thawed the cDNA twice (maximum), can they degrade this quickly when stored at -20? Or have I contaminated them, and what can I do to avoid this in the future?

Also, my PI is not helpful on this. How do you do PCR figures (like bands on a gel picture)?

Thank you!


#2 Mad Researcher

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Posted 13 June 2015 - 12:27 PM



Did you keep the qPCR plate at -20, thawed it and analyzed again? Maybe the products have started to degrade. I keep my plates at 4 degrees and try to analyze them on gel (if i have doubts or just want to be double check) within a week. Keeping the products for too long may degrade.


I don't think freeze thawing cDNA would degrade it. I have been thawing my samples but i still get my results. How were your Ct values. Were they the same as before? Also, What about your positive and negative controls? Did they show any signs of contamination?


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#3 phage434



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Posted 14 June 2015 - 07:14 AM

DNA is not particularly stable in a PCR buffer, although it is unlikely for it to degrade when frozen. If you plan on keeping it around, adding EDTA will chelate magnesium and inhibit DNAse activity. Something like a 50x dilution of the 500 mM EDTA stock solution into your DNA. This may affect downstream application of enzymes (restriction digestion or PCR e.g.) so be aware of that.

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