DNA recovery

Jeremy Murray bmbjmm at bmb.leeds.ac.uk
Sun Sep 27 06:42:12 EST 1998

Patrick F.H. Lai the Graduate Student wrote:
> ---- Begin Original Message ----
> Hi, do you have any ideas on DNA recovery from agarose gel ?
>  I'm doing in vitro transcription and need large amount of high quanlity
> DNA template (DNA should be in 10 to 20 microgram) without any RNase or RNA
> contamination.
>  To prepare DNA template, I usually did miniprep, linearized miniprep DNA
> by restriction enzyme digestion, run low melting agarose gel and purified
> DNA from LMT gel, I found the recovery of linearized DNA was very low. you
> might have experience on in vitro transcription or prepare DNA template for
> in vitro synthesis of RNA, how did you get relative large amount of DNA ?
> thanks
> ---- End Original Message ----
> Dear Jing Zhang and Netters,
> This is what I do when I prepare templates for in vitro transcription:
> I usually digest a portion of a MaxiPrep, because the [DNA] is higher than what I can get with a MiniPrep (unless I lyophilise the MiniPrep and reconstitute in a smaller volume.... tried this as well, it works the same.)
> I try to use strong enzymes, like EcoRI and HindIII, but not weak and fragile ones like PvuI.  Sometimes, there is no choice, though.
> I would try to buy enzymes that are concentrated, or use as much enzyme as possible, bearing in mind that too much glycerol from the enzyme storage buffer inhibits the digestion.
> I usually digest at 37 deg C for at least 18 hours, sometimes 24 hours if the enzyme is old or did not cut that nicely the previous time.  I don't worry about star activity because I think there is enough DNA from a MaxiPrep to avoid over-digestion.  Avoiding enzymes with star activity is not a bad idea, though.
> After digestion, I would clean up the DNA using GeneClean II (or III) from Bio101, lyophilize the (twice) eluted DNA and resuspend the dried DNA in RNase-free Tris buffer (which comes with GeneClean III).  I think DNA recovery is better than doing gel extraction.
> A small portion of the DNA is then electrophoresed in an agarose gel with the uncut MaxiPrep in the next lane.
> If I see a single strong band migrating slightly slower than the uncut supercoiled MaxiPrep DNA, I would take what I have prepared as completely linearized DNA and use it in in vitro transcription.
> Otherwise, I would repeat the digestion, possibly with more or new enzymes.(Obviously, if the enzyme cuts more than once, this does not apply.)
> [DNA] is guesstimated by comparing intensity with the bands of a lambda-HindIII Marker in the EtBr-stained gel.
> I usually use about 5% of a MaxiPrep and got enough cut DNA for about 10 to 20 small-scale in vitro transcription reactions with hot UTP, that is if there is a single band of linearized template.
> This also is enough for a handful of large scale cold RNA synthesis.
> Note :  GeneClean III is a good kit, but I am thinking of switching to Qiagen's QIAquick Spin columns for ease of use, as I sometimes have to prepare 10 templates together.
> Comments, anyone ?
> Hope this is useful info.   :-)
> Patrick F.H. Lai  < PFHLai at looksmart.com >
> Graduate Student
> University of Toronto
> Toronto, Ontario, Canada
> P.S.  I am merely a GradStudent.
> One should take a HUGE grain of salt with whatever I have typed above.
> Good Luck.   :-)
> LookSmart ? or keep looking.
> http://www.looksmart.com

my experince wrt DNA recovery post restriction
is that the QiaQuick spin columns are the best
we have used
for speed, convenience, and yield

the only bad thing is that if you are doing 
a lot of cloning etc
they add quite a bit to the lab consumables budget
but they make the lab more productive

wrt restriction digests
i donot thingk there is much to be gained by incubation times >2hrs
check the activity of the enzymes you are using in manuals such as NEB
all enzymes are subjected to rigorous QC checks
some are better than others
hope this helps
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Jeremy Murray                           MAIL: bmbjmm at bmb.leeds.ac.uk
Biochemistry & Molecular Biology        TEL:  +44 113 233 2591
Leeds University, LEEDS LS2 9JT 	FAX:  +44 113 233 3167
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