DNA recovery

Patrick F.H. Lai the Graduate Student pfhlai at LOOKSMART.COM
Sun Sep 27 16:46:19 EST 1998

---- Begin Original Message ----
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:

> 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 ?
> <snip>

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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Biochemistry & Molecular Biology        TEL:  +44 113 233 2591
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Dear Jez and Netters,

I think restriction digestions done to linearize plasmids for the purpose of preparing templates for in vitro transcription may be an exception -- long incubation helps.

One does not want to have unlinearized plasmid templates in the in vitro transcription reaction, or the RNA polymerase would go round and round the circular plasmid --- God knows when it may fall off --- quickly depleting nucleotides without synthesizing enough of the intend RNA product.  I usually linearize my plasmids overnight so that there is less chance of getting an incomplete digestion.

The alternative would be doing a gel extraction.  The draw back would be low recovery of DNA, which is the problem that started this thread by Jing Zhang.

With respect to DNA purification columns, has anyone tried the new stuffs from GIBCO BRL / Life Technologies ?  They have a new product line named "Concert".  The stuffs seem like Qiagen columns on screen: < http://www2.lifetech.com:80/catalog/techline/molecular_biology/product_description/concert.html >.  
A sales rep said that they are 10~15% cheaper than Qiagen's, but their performance is at least as good.  

Has anyone used CONCERT yet ?  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.   :-)


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