Size Fractionation Using Agarose Gels

Thomas Quertermous1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 5.4
DOI:  10.1002/0471142727.mb0504s34
Online Posting Date:  May, 2001
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Abstract

In the , digested genomic DNA is size fractionated on a slab agarose gel, and the appropriate region of the gel is defined by Southern blot analysis (for subgenomic libraries) or by size (for genomic libraries) and the DNA is eluted. An describes electrophoresis using a Bull's‐eye apparatus. Digested genomic DNA is loaded onto a large preparative circular agarose gel in this apparatus, the DNA fragments are electrophoresed toward the center of the gel and then eluted. Fragments leaving the gel are pooled and constitute a fraction. Fractions containing the gene of interest are identified by Southern blotting of a small aliquot of alternate fractions. This procedure allows the purification of large amounts of size‐fractionated DNA that is particularly well suited for genomic library construction and normally allows creation of large numbers of recombinant clones.

     
 
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Table of Contents

  • Basic Protocol 1: Electrophoresis on a Slab Agarose Gel
  • Alternate Protocol 1: Electrophoresis on the Bull's‐Eye Agarose Gel Apparatus
  • Commentary
     
 
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Materials

Basic Protocol 1: Electrophoresis on a Slab Agarose Gel

  Materials
  • 400 µg to 2 mg digested genomic DNA (unit 5.3)
  • DNA size markers
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5), Southern blot transfer (unit 2.9), ethanol precipitation (unit 2.1), and hybridization (units 2.9 & 6.3)

Alternate Protocol 1: Electrophoresis on the Bull's‐Eye Agarose Gel Apparatus

  Additional Materials
  • TAE buffer ( appendix 22)
  • Chloroform
  • 0.5 M EDTA, pH 7.5
  • Buffered phenol (unit 2.1)
  • TE buffer ( appendix 22)
  • Bull's‐eye electrophoresis apparatus ( Hoefer)
  • Bull's‐eye electronic control apparatus ( Hoefer)
  • Dialysis tubing (flat width >1.69 in.)
  • Peristaltic pump, fixed rate (20 ml/min)
  • Peristaltic pump, variable rate
  • Fraction collector (capable of holding 16 × 150 mm tubes; fraction time adjustable up to 1.5 hr)
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Figures

Videos

Literature Cited

Literature Cited
   Bott, F., Moran, C.P., Edgell, M.H., Drew, B., and Charles, L. 1980. Direct fractionation of genes by preparative electrophoresis of Bacillus subtilis DNA. Gene 10:283.
   Carreira, L.H., Carlton, B.C., Bobbio, S.M., Nagao, R.T. and Meagher, R.B. 1980. Construction and application of a modified “Gene Machine”: A circular concentrating preparative gel electrophoresis device employing discontinuous elution. Anal. Biochem. 106:455‐468.
   Edgell, M.H., Weaver, S., Haigwood, N., and Hutchinson, C.A. 1979a. Gene enrichment. In Genetic Engineering, Vol. I (J.K. Setlow and A. Hollander, eds.) pp. 37‐49. Plenum, New York.
   Polsky, F., Edgell, M.H., Seidman, J.G., and Leder, P. 1978. High‐capacity gel preparative electrophoresis for purification of fragments of genome DNA. Anal. Biochem. 87:397‐410.
   Southern, E.M. 1979a. Gel electrophoresis of restriction fragments. Meth. Enzymol. 68:152‐176.
   Southern, E.M. 1979b. A preparative gel electrophoresis apparatus for large‐scale separations. Anal. Biochem. 100:304.
Key Reference
   Southern, E.M. 1979a. See above.
  Describes construction of a Bull's‐eye electrophoresis apparatus and principles that govern separation of DNA fragments on this device.
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