PFGE

John Nash nash at biologysx.lan.nrc.ca
Thu Jun 10 10:45:04 EST 1993


In article <930610123217.503 at cbr.dwe.csiro.au> MPB at CBR.DWE.CSIRO.AU (Mark Bradley) writes:
>From: MPB at CBR.DWE.CSIRO.AU (Mark Bradley)
>Subject: PFGE
>Date: 10 Jun 93 17:32:17 GMT

I was the one who asked the original question.  Thanks to the many who 
replied - I am collecting the responses, and will post a summary on Monday.


>Re how does one make plugs?
>From Wafa.el-adhami at anu.edu.au" who read this request but is
>not on the network. Please contact her directly for more information.


>....."We think that the best way of making
>plugs is to use a home-made mould designed to fit the teeth of a comb to be
>used for electrophoresis.  The best suitable dimensions we find useful and
>fit the combs we use are those of 10mmx8mmx2mm (width x hight x thickness),
>each one rectangle of these contains 2 g of DNA which can be easily cut
>into two halves across the hight to give an agarose plug with 10mmx4mmx2mm
>that can nicely and snugly eased into the wells of the running gels.  This
>ensures a very good PFGE gel because one gets lanes of cut DNA with sharp
>bands of 10mm wide (we certainly get great gels from both FIGE and CHEF).
>We have recently sent for publication a nice paper on plug making (chapter)
>to Methods in Gene Technology (in press).
>One other method that we have come across (K. Matthaei) is to squirt small
>volumes of the agarose slurry mixed with cells onto a parafilm piece (it is
>best to use small volumes ~<50 l).  Because of surface tension the agarose
>will form half spheres that can be collected and treated for lysis, etc.
>However, when these are loaded one does not get wide nicely spread bands of
>cut DNA; others melt there plugs after digestion, but we are unsure how
>that might affect large fragments (~800kb, sheering, etc!) we have not
>tested that effect.  But we do melt plugs digested with frequent cutters
>and get good gels too." 

I have a plug mould like Wafa suggested (but only 1 mm thick - my well 
thickness), but my plugs keep on ripping when I scoop them them from my 
lysis --> wash --> re digest.  I'm using a half volume of 1.5% LMT agarose 
in TE (plus a half volume of cell suspension).  My mould can be doubled up 
to make plugs twice as thick, and I can do the same with my wells... I 
suppose I should try that, or should I just be more careful?

Thanks again, (and Wafa - I know I still owe you an email, but I haven't run 
a nice enough gel yet).


  cheers, John

  John Nash                           | Email: Nash at biologysx.lan.nrc.ca.
  Institute for Biological Sciences   |
  National Research Council of Canada | Email to my other NRC accounts
  Ottawa, Ontario, Canada.            | is usually forwarded here.
	  *** Disclaimer:  All opinions are mine, not NRC's! ***



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