Cloning PCR products

Keith Rand Keith.Rand at molsci.csiro.au
Mon Oct 27 23:39:34 EST 1997


In article <3454D28A.4565 at Aptagen.com>, Geoffrey Kidd <GKidd at Aptagen.com> wrote:

> One factor to watch out for is U.V. exposure.  A large plasmid such as 
> yours makes a larger target for U.V. damage than do the 2-3 kb 
> varieties.  Be sure to block as much U.V. as possible when excising from 
> the gel, allowing just enough fluorescence to see your DNA, and excise 
> it as quickly as possible.  I found that our light box was able to 
> destroy all of a 3 kb plasmid after about 5-6 minutes. 
> 
> As a control to see if this is a problem, just try re-ligating some cut 
> plasmid, without insert, before and after gel-purification.


For several years now I've avoided any UV exposure of my precious DNA
fragments by running gels with crystal violet in both gel and buffer.
Usually I can see the bands as they separate. If there's not much DNA then
one can do a quick stain and destain (around 30mins) after running the
gel. I certainly feel that all of my cloning work is a lot more consistent
since avoiding the transilluminators. And if you have to use a
transilluminator it may be best to check that nobody has put shorter
wavelength bulbs in it in order to see their bands more easily - I know of
this happening! 

The crystal violet paper was written up in the Technical tips Online (Elsevier)

Free registration: http://tto.trends.com/cgi-bin/tto/pg_reg.cgi

Then I think 

http://tto.trends.com/cgi-bin/tto/pr/pg_art.cgi?sid=CAT3&ac=t40022|/cgi-bin/tto/pr/pg_cat.cgi?cc=CAT3

should get you to the crystal violet paper (you'd better cut and paste that!)

-- 
Keith Rand,  Sydney Australia



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