PFGE - agarose plugs (summary)

John Nash nash at biologysx.lan.nrc.ca
Mon Jun 14 12:47:10 EST 1993


In article <nash.169.0 at biologysx.lan.nrc.ca> I wrote:

>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.

Here is the promised summary:

Many thanks to all of you who replied.  Score <one> for bionet!!!  So many 
suggestions, so little time!  Replies were edited in the following way - 
line wrap adjusted for my mailer --> hope it works for yours; headers 
and .signatures removed.  
---------------- clip --------------------------------------------------

From: TLANE at watson.princeton.edu

   What I did is take some dental modelling compound and made a cast of the 
comb that I was using (laid flat side down) so it formed trougths the width
and length of the comb and about as deep as the comb is thick.  I pipetted 
the molten agarose into one end of the trough and when it solidified I used 
a filed down small spatula to manipulate the plugs. I think that this system 
worked very well and it was cheap.

==============================================================================
From: wjb1 at kimbark.uchicago.edu (William J. Buikema)

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.

==============================================================================
From: MPB at CBR.DWE.CSIRO.AU (Mark Bradley)



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