DNA fragment isolation
svetlov at oncology.wisc.edu
Tue Dec 2 15:10:27 EST 1997
In article <Pine.SOL.3.96.971201162508.22341A-100000 at apocalypse>, "Rachel
N. Fish" <rfish at ocf.Berkeley.EDU> wrote:
> I am making DNA templates by PCR amplification to be used in
> transcription. I have two major concerns. 1) The effects of UV on my
> fragment. UV has been shown to have an effect on transcription. Hence, I
> would like to minimize (or do away with!) such exposure. 2) High
> efficiency recovery. Since these templates are a major part of my
> research, I would like to make enough to last for awhile.
> After running my PCR samples on an agarose gel, I have tried
> electroelution, QIAgen, GeneClean, all with varying (and sometimes very
> low) results.
> Does anyone have any suggestions? If this question has already been
> answered, I apologize for the repeat, but I'm new to this group. I tried
> searching the archives but didn't come up with anything quite appropriate.
> Thank you in advance for your assistance.
> -Rachel Fish
> rfish at ocf.berkeley.edu
UV - there are few things you can try, such as using long wavelength UV
with EtBr stained gel (you can also use 300 nm with the SYBR stain but this
shit is expensive). I also use polycarbonate support for the gel (that
casting tray) or a glass sheet to filter out some UV. Somebody told me that
they've cured the nicks in the DNA by incubating the purified DNA with low
conc. T4 DNA ligase/ATP but I never was that desperate.
As for yields in elution my experience with Qiagen was similar to yours and
I don't use Geneclean often and not for quantitative recovery. I'm firm
believer in DE81 paper and everybody who saz he can't make it work should
be ashamed of thimself. Recovery I'm getting is consistently 70-80% of
input, just don't forget to smash the paper into pulp when eluting.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
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