HELP!!! In-efficient Ligations? (Long)

Ned Mantei mantei at cell.biol.ethz.ch
Mon May 8 13:10:30 EST 2000


In article <3916ea6e$1 at news.sbg.ac.at>, "news.sbg.ac.at" 
<comobis1 at natur.sbg.ac.at> wrote:

>I´ve solved compareable problems in our lab in the following way:
>Minimize the time on the UV transilluminator !
>Hide the bands on the gel, you are not working on, with several layers of
>underlaying thick aluminium foil.
>And again: Shorten the time on the UV.


There was an article on this subject some years ago in BioTechniques. 
The authors applied DNA solutions to the slots of an agarose gel and 
measured remaining infectivity as a function of time on the UV box. With 
312 nm wavelength UV, a plasmid of something like 5 kb was 99% dead 
within 1 min. The longer the plasmid the greater the UV 
sensitivity,(which would also fit with the observations of the original 
poster, Mark Youles). It helped to keep the gel on the plastic tray, but 
not all that much. The long-term solution was to get a UV box emitting 
at 360 nm. With this the authors showed that you can leave the gel on 
the box for more than 10 minutes without any appreciable loss in 
infectivity.

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland




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