Size Fractionation Using Sucrose Gradients

John H. Weis1, Thomas Quertermous2

1 Harvard Medical School, Boston, Massachusetts, 2 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 5.3
DOI:  10.1002/0471142727.mb0503s00
Online Posting Date:  May, 2001
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Abstract

DNA fragments migrate through a linear sucrose gradient at a rate that is dependent on their size. This procedure described in this unit provides good resolution for DNA fragments 5 to 60 kb in size, and sucrose gradients are also useful for purification of bacteriophage l vector arms. Partially digested genomic DNA can be fractionated for the production of cosmid or bacteriophage libraries and completely digested DNA can be fractionated for subgenomic DNA libraries. Protocols are provided in this unit for both partial and complete enzyme digestion of genomic DNA.

     
 
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Table of Contents

  • Section II: Preparation of Insert DNA From Genomic DNA
  • Basic Protocol 1: Sucrose Gradient Preparation of Size‐Selected DNA
  • Support Protocol 1: Partial Enzyme Digestion
  • Support Protocol 2: Complete Enzyme Digestion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Sucrose Gradient Preparation of Size‐Selected DNA

  Materials
  • Completely or partially digested genomic DNA (support protocols)
  • STE buffer ( appendix 22)
  • recipe10% and recipe40% sucrose solution
  • 0.9% agarose gel
  • 100% ethanol
  • TE buffer ( appendix 22)
  • Sucrose gradient maker
  • Beckman SW‐28 or SW‐41 rotor or equivalent
  • Additional reagents and equipment for ethanol precipitation (unit 2.1) and agarose gel electrophoresis (unit 2.5)

Support Protocol 1: Partial Enzyme Digestion

  Additional Materials
  • High‐molecular‐weight genomic DNA (units 2.2, 2.3 & 2.4)
  • 10× restriction enzyme buffer (unit 3.1)
  • recipeStop solution
  • Restriction enzyme for DNA digestion (unit 3.1)
  • 0.5% agarose gel (unit 2.5)
  • DNA size markers (unit 2.5)
  • 1:1 phenol/chloroform (unit 2.1)
  • 5 M NaCl
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Figures

Videos

Literature Cited

Literature Cited
   Seed, B., Parker, R.C., and Davidson, N. 1982. Representation of DNA sequences in recombinant DNA libraries prepared by restriction enzyme partial digestion. Gene 19:201‐209.
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