Patrick F.H. Lai the Graduate Student
pfhlai at LOOKSMART.COM
Sat Sep 26 11:39:04 EST 1998
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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
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 ?
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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 >
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. :-)
or keep looking.
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