Efficient method for analysis of recombinants?

Chris Boyd chrisb at hgu.mrc.ac.uk
Thu Jan 30 05:19:21 EST 1997

Alexander N. Kukushkin (kan at mmcd.cyt.ras.spb.ru) wrote:
: >   Rapid analysis of plasmids containing the insert can be done (according
: >   to Maniatis) by resuspending the colonies in SDS, heating them and load
: >   them into an agarose gel without ethidium bromide. After
: >   electrophoresis the staining with EtBr should be done to visualise the
: >   DNA and select the recombinants.
: >
: >   My question is: does this actually work even with small inserts (i.e.
: >   300bp)?
: >
: >   Will Garcia-Asua
: >   mbp95gg at sheffield.ac.uk
: >
: These small inserts could be revealed by 0,7-1% agarose gel for long running.
: Alex

Yes, but remember you are looking at supercoiled circular DNA, so
standard size markers will not be even remotely suitable.  Your size
standards have at the very least to be (i) the original plasmid vector
and (ii) a plasmid of the size you are looking for. The method is only
suitable as a preliminary screen for "needle in a haystack" type
experiments.  For standard clonings, minipreps + digestion is quicker
and more informative.

Best wishes,
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND

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