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I am trying to do some DNA extraction for my professor, and I make a lysis buffer with proteinase K. I did not have the time to continue the procedure so the hair and the buffer have been sitting together at room temperature for about 12 days. My professor stated it would be "fine" to continue on with my DNA isolation procedure, but I have my doubts.

Should I continue my DNA extraction( vortexing, boiling, centrifugation and collection of supernatant, storage at -20 degree Celsius) or would this be pointless at this time? My goal is to run PCR and then a gel.

EDIT:

Here is the composition of the lysis buffer:

In 20 ml of Lysis buffer:

final concentrations are $0.1 M$ TAE, $0.5M$ $\ce{NaCl}$, 0.2% SDS.

To $800 ul$ of Lysis buffer I added $5 ul$ of proteinase K at a concentration of $250 ug/ml$.

(Proteinase K was made via: $ 0.0025 g$ up to $10 ml$ of TAE buffer( $1 M $))

This was in a $1.5 ml$ centrifuge tube that has been now sitting for 12 days.

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    $\begingroup$ Can you put in the composition of the lysis buffer here? $\endgroup$ – Chris Mar 27 '16 at 18:49
  • $\begingroup$ @Chris I put the composition of the buffer, thanks for reminding me. $\endgroup$ – Ro Siv Mar 27 '16 at 21:51
  • $\begingroup$ What do you mean by 0.1M TAE? TAE normally stands for Tris-Acetate-EDTA, but you already have EDTA as a component. $\endgroup$ – March Ho Mar 28 '16 at 23:46
  • $\begingroup$ @MarchHo Yea your right, I was not thinking sorry let me fix that. $\endgroup$ – Ro Siv Mar 29 '16 at 0:10
  • $\begingroup$ @rosiv Hi Ro, I wanted to know if your PCR was succesfull. I am planning to Transport WORM samples in Lysis Buffer at ambient temperature from Asia to Germany, do you have any suggestions for me?? Thanks! $\endgroup$ – user24150 May 28 '16 at 6:16
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It should be fine. Unless they have been contaminated with DNase, none of the components will damage or denature DNA. I've left purified plasmid DNA on the bench for weeks with no apparent degradation, so you should be okay (not that I recommend doing that on a regular basis). I would continue with the purification, according to your professor's recommendation, because you've already come this far. If worst comes to worst, you won't get anything, and you'll have to re-purify. However, the chances of your getting something are pretty high, so you may as well go with it.

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  • $\begingroup$ Ironically I tried to continue my extraction and failed spectacularly, so I might have to redo the sample collection. This will help for the future though. $\endgroup$ – Ro Siv Mar 29 '16 at 0:11
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Your concentration of EDTA seems to be rather high. When performing lysis for DNA extraction, I usually use 1mM EDTA in the lysis buffer. Concentrations that are too high may result in inhibition of the PCR reaction later on, unless the DNA is first purified.

This could be a reason why your reaction failed.

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  • $\begingroup$ I had less EDTA than that, the EDTA I mentioned previously was the final concentration of EDTA in my TAE, I removed it because I was not thinking clearly sorry. The final concentration of my EDTA in my TAE is 0.025 M i believe. Also I have not ran my reaction yet, but many thanks for the advice. $\endgroup$ – Ro Siv Mar 30 '16 at 3:49
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    $\begingroup$ @rosiv how then did you know your reaction failed? Boiling the mixture should result in precipitation of proteins, which should be removed by the spin. You will be left with the DNA solution which you can either use directly or use after desalting. $\endgroup$ – March Ho Mar 30 '16 at 3:52

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