Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels

David Moore1, Joanne Chory2, Randall K. Ribaudo3

1 Massachusetts General Hospital, Boston, Massachusetts, 2 The Salk Institute, La Jolla, California, 3 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.5
DOI:  10.1002/0471142735.im1005s08
Online Posting Date:  May, 2001
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Abstract

This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first basic protocol describes electroelution of the fragment of interest from standard agarose gels using buffer‐filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA‐45 paper is also described and provides relatively high yields for fragments smaller than 2000 bp. Protocols are also provided for separating fragments larger than 1000 bp using low gelling/melting agarose gels and purified by phenol extraction, b‐agarase digestion of the gel, or via glass beads extraction. Sieving agarose gels can also be used to resolve very small DNA fragments. Removing linkers from a fragment using a column rather than a gel is included, followed by a method for estimating DNA concentrations in solution.

     
 
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Table of Contents

  • Basic Protocol 1: Electroelution from Agarose Gels
  • Basic Protocol 2: Electrophoresis onto NA‐45 Paper
  • Basic Protocol 3: Isolation of DNA Fragments Using Low Gelling/Melting Temperature Agarose Gels
  • Alternate Protocol 1: Recovery of DNA from Low Gelling/Melting Temperature Agarose Gels Using β‐Agarase Digestion
  • Alternate Protocol 2: Recovery of DNA from Low Gelling/Melting Temperature Agarose Using Glass Beads
  • Alternate Protocol 3: Purification Using Sieving Agarose Gels
  • Alternate Protocol 4: Removal of Oligonucleotide Fragments Using a Sephacryl S‐300 Column
  • Support Protocol 1: Rapid Estimation of DNA Concentration by Ethidium Bromide Dot Quantitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Electroelution from Agarose Gels

  Materials
  • DNA to be eluted
  • Appropriate restriction enzymes and buffers (unit 10.8)
  • Ethidium bromide solution (unit 10.4)
  • TAE buffer (unit 10.4)
  • recipeElutip high‐salt solution
  • 2.5 M NaCl
  • recipeElutip low‐salt solution
  • 100% and 70% ethanol
  • TE buffer, pH 8.0 ( appendix 2A)
  • Spectrapor 3 dialysis membrane tubing (11.5‐mm diameter with molecular‐ weight‐cutoff of 3500; American Scientific #01614‐21)
  • Elutip‐d columns (Schleicher & Schuell)
  • Small syringe (e.g., 5‐ml)
  • Additional reagents and equipment for restriction enzyme digestion (unit 10.8) and agarose gel electrophoresis and photography (unit 10.4)

Basic Protocol 2: Electrophoresis onto NA‐45 Paper

  Materials
  • Ultrapure agarose (e.g., SeaKem GTG agarose, FMC Bioproducts)
  • NA‐45 paper (Schleicher & Schuell)
  • TE buffer, pH 8.0 ( appendix 2A)
  • recipeNA‐45 elution buffer
  • Buffered phenol (unit 10.1)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 10.1)
  • 95% and 70% ethanol, ice cold
  • Flat forceps, two pairs
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4) and DNA extraction and precipitation (unit 10.1)

Basic Protocol 3: Isolation of DNA Fragments Using Low Gelling/Melting Temperature Agarose Gels

  Materials
  • DNA to be isolated
  • Appropriate restriction enzymes and buffers (unit 10.8)
  • Low gelling/melting temperature agarose (SeaPlaque; FMC Bioproducts)
  • Ethidium bromide solution (unit 10.4)
  • TE buffer, pH 8.0 ( appendix 2A)
  • Buffered phenol (unit 10.1)
  • Elutip recipehigh‐salt and recipelow‐salt solutions
  • Scalpel
  • Elutip‐d column (Schleicher & Schuell)
  • Beckman JS‐13 swinging‐bucket rotor (or equivalent)
  • Additional reagents and equipment for restriction enzyme digestion (unit 10.8), agarose gel electrophoresis (unit 10.4), and ethanol precipitation (unit 10.1)

Alternate Protocol 1: Recovery of DNA from Low Gelling/Melting Temperature Agarose Gels Using β‐Agarase Digestion

  Additional Materials
  • β‐agarase I (New England Biolabs #392S or Calbiochem #121820)
  • recipeβ‐agarase buffer
  • Additional reagents and materials for dialysis ( appendix 3A) and isopropanol precipitation of DNA (unit 10.1)

Alternate Protocol 2: Recovery of DNA from Low Gelling/Melting Temperature Agarose Using Glass Beads

  Additional Materials
  • NaI solution (unit 10.1)
  • Glass beads suspension (unit 10.1)
  • 15‐ml polypropylene tube
  • Additional reagents and equipment for glass beads purification of DNA (unit 10.1)
NOTE: The materials required for this procedure are also provided in commercial kits (e.g., GeneClean, Bio101; Glas‐Pac, National Scientific Supply; and Qiaex Gel Extraction kit, Qiagen).

Alternate Protocol 3: Purification Using Sieving Agarose Gels

  Additional Materials
  • 50% (v/v) Sephacryl S‐300 (Pharmacia Biotech) in TE buffer, pH 8.0 (Sephacryl/TE), stored covered at 4°C
  • 0.3 M NaCl in TE buffer, pH 8.0 (NaCl/TE)
  • DNA sample in NaCl/TE solution
  • Glass wool plug and 6‐in. Pasteur pipet, siliconized ( appendix 3A)

Alternate Protocol 4: Removal of Oligonucleotide Fragments Using a Sephacryl S‐300 Column

  Materials
  • DNA: purified DNA for standards and isolated DNA to be quantitated (previous protocols)
  • TE buffer, pH 8.0 ( appendix 2A)
  • 1 µg/ml ethidium bromide (store wrapped in foil at 4°C)
  • Plastic wrap
  • UV transilluminator
  • Additional reagents and equipment for gel photography (unit 10.4)
CAUTION: Ethidium bromide is a mutagen and should be handled with care.
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Figures

Videos

Literature Cited

Literature Cited
   Blin, N., Gabain, A.V., and Bujard, H. 1975. Isolation of large molecular DNA from agarose gels for further digestion by restriction enzymes. FEBS Lett. 53:84‐86.
   Chory, J. 1987. Nondenaturing polyacrylamide gel electrophoresis. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, eds.) pp. 2.5.9‐2.5.15. Greene Publishing and Wiley‐Interscience, New York.
   Vogelstein, B. and Gillespie, D. 1979. Preparative and analytical purification of DNA from agarose. Proc. Nat. Acad. Sci. U.S.A. 76: 615‐619.
   Wienand, U., Schwarz, Z., and Felix, G. 1978. Electrophoretic elution of nucleic acids from gels adapted for subsequent biological tests: Application for analysis of mRNAs from maize endosperm. FEBS Lett. 98:319‐323.
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