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Question about DNA purification from a restriction digestion reaction

restriction digest DNA PCR purification heat inactivation AvrII

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#1 djhz2001

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Posted 12 September 2013 - 03:33 PM

Hello,

 

I would like to receive some feedback from people who have some experience with restriction digestion and DNA purification.

 

I set up a digestion of a plasmid that is 8 kb in size. I digested 3 ug of the plasmid in a 40 ul Rx, using 12 units of AvrII restriction enzyme (cuts my plasmid only once). I ran the Rx O/N. Then, I proceeded to run 2 ul of the digest in a 1% gel in order to see that my DNA was cut (uncut DNA will run much lower than my 8 kb marker; I also ran an uncut control in a different occasion which confirmed that the uncut plasmid ran faster in a 1% gel). My gel picture showed me a clear sharp band at 8 kb with no smears or anything else that would indicate star activity or DNA degradation, so I was confident that my DNA was properly cut.

 

After this, I proceeded to purify my cut DNA using a PCR purification kit without heat inactivation of the enzyme. Previously, this same procedure has yielded in 50 - 60 % DNA recovery in the past when I used StuI restriction enzyme (also a single cutter for this plasmid) instead of AvrII. However, when I applied the same procedure to the AvrII cut DNA, my recovery was of 10%, which gave me insufficient DNA for subsequent experiments.

 

I would like to know if anyone in this forum has experienced something like this. I re-measured the DNA concentration from the purified DNA, as well as the original uncut plasmid stock, and it always gave me the same DNA concentrations. More specifically, I would like to know if anyone here knows whether heat inactivating a restriction enzyme will increase the DNA recovery.

 

Thank you all for your time.



#2 phage434

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Posted 12 September 2013 - 05:33 PM

I don't know if this will affect recovery, but I would certainly suggest heat killing the enzymes whenever possible. AvrII can be killed at 80C, but the BSA in new NEB buffers stabilizes the enzyme. You may want to digest in a buffer without BSA. We do digests in a cycler, so the heat kill is just a step in the cycler program. I would also recommend that your digestion volume should be larger. Likely the majority of your volume is DNA sample, which often contains reaction inhibitors. Diluting those will make the reaction work better. You likely also do not need an overnight digestion -- most of it will happen in the first 15 minutes.







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