PFGE agarose plugs

William J. Buikema wjb1 at kimbark.uchicago.edu
Thu Jun 10 09:05:34 EST 1993


In article <nash.169.0 at biologysx.lan.nrc.ca> nash at biologysx.lan.nrc.ca (John Nash) writes:
>Hi..
>
>What do people use for agarose plug formers when they are embedding cells in 
>agarose prior to lysis, restriction digests, etc for PFGE?  Do I have to 
>go out and buy an expensive mould?  I got tech. support to make a rather 
>nice mould, such that the plugs will fit in my gel wells, but the plugs are 
>1 mm thin and rather brittle.  Should I perservere, or is there a nice trick 
>with a syringe, or something?
>
>If you email replies to me, I'll summarize and post.
>
>
>  cheers, John

An alternative to making agarose plugs is to make agarose beads.  The technique
is simple: add your washed cells to LMP agarose, then add this still liquid
mixture to ice cold paraffin oil and shake.  The oil is easily removed, and
the beads can be made to almost any size by how hard one shakes.  Plus the
beads can be pipetted easily and washed quickly.  For the full procedure,
check out:

Kuritz, T., A. Ernst, T. A. Black, and C. P. Wolk. 1993. High-resolution 
mapping of genetic loci of Anabaena PCC 7120 required for photosynthesis 
and nitrogen fixation. Mol. Microbiol. 8:101-110.


William J. Buikema              internet: wjb1 at midway.uchicago.edu
University of Chicago               or    w-buikema at uchicago.edu
Dept. of Molecular Genetics &       or    hetman at uchicago.edu 
    Cell Biology, CLSC 313
920 E. 58th St.
Chicago, IL  60637



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