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)?
cDNA degradation within a week at -20?
Posted 12 June 2015 - 12:25 PM
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?
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.