Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels

David Moore1, Dennis Dowhan1, Joanne Chory2, Randall K. Ribaudo3

1 Baylor College of Medicine, Houston, Taxas, 2 The Salk Institute, La Jolla, California, 3 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.6
DOI:  10.1002/0471142727.mb0206s59
Online Posting Date:  August, 2002
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Abstract

This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first 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 (second protocol) provides relatively high yields for fragments <2000 bp. Fragments >1000 bp can also be separated on low gelling/melting agarose gels and purified by phenol extraction (third protocol), b‐agarase digestion of the gel (first alternate protocol), or via glass beads extraction (second alternate protocol). Removing linkers from a fragment using a column rather than a gel is described, 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 Agarose Using Silica Membrane Spin Columns
  • Alternate Protocol 3: 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 encoding sequence of interest
  • Appropriate restriction enzymes and buffers (unit 3.1)
  • Ethidium bromide solution (unit 2.5)
  • TAE buffer ( appendix 22)
  • recipeElutip high‐salt solution (see recipe)
  • 2.5 M NaCl
  • recipeElutip low‐salt solution (see recipe)
  • 100% and 70% ethanol
  • TE buffer, pH 8.0 ( appendix 22)
  • Spectrapor 3 dialysis membrane tubing (11.5‐mm diameter with MWCO of 3500; Baxter)
  • Elutip‐d columns (Schleicher & Schuell)
  • Small syringe (e.g., 5‐ml)
  • Additional reagents and equipment for restriction enzyme digestion (unit 3.1) and agarose gel electrophoresis and photography (unit 2.5)

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 22)
  • recipeNA‐45 elution buffer (see recipe)
  • Buffered phenol (unit 2.1)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 95% and 70% ethanol, ice cold
  • Flat forceps, two pairs
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and DNA extraction and precipitation (unit 2.1)

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 3.1)
  • Low gelling/melting temperature agarose (SeaPlaque; FMC Bioproducts)
  • Ethidium bromide solution (unit 2.5)
  • TE buffer, pH 8.0 ( appendix 22)
  • Buffered phenol (unit 2.1)
  • recipeElutip high‐salt and recipelow‐salt solutions (see reciperecipes)
  • Scalpel
  • Elutip‐d column (Schleicher & Schuell)
  • Beckman JS‐13 swinging‐bucket rotor (or equivalent)
  • Additional reagents and equipment for restriction enzyme digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), and ethanol precipitation (unit 2.1)

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

  • β‐agarase I (New England Biolabs or Calbiochem)
  • recipeβ‐agarase buffer (see recipe)
  • Additional reagents and materials for dialysis ( appendix 3D) and isopropanol precipitation of DNA (unit 2.1)

Alternate Protocol 2: Recovery of DNA from Agarose Using Silica Membrane Spin Columns

  • 6.0 M NaI solution (filter through filter paper, store up to 3 months in the dark at 4°C)
  • recipeBinding buffer (see recipe)
  • Wash buffer (unit 2.1)
  • TE buffer, pH 8.0 ( appendix 22) or nuclease‐free H 2O
  • 1.5‐ml microcentrifuge tubes
  • 45° to 50°C water bath
  • Silica membrane spin columns (e.g., Qiagen, Promega, Invitrogen, Novagen)

Alternate Protocol 3: Removal of Oligonucleotide Fragments Using a Sephacryl S‐300 Column

  • 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 solution)
  • DNA sample in NaCl/TE solution
  • Glass wool plug and 6‐in. Pasteur pipet, silanized ( appendix 3B)

Support Protocol 1: Rapid Estimation of DNA Concentration by Ethidium Bromide Dot Quantitation

  Materials
  • DNA: purified DNA for standards and isolated DNA to be quantitated (previous protocols)
  • TE buffer, pH 8.0 ( appendix 22)
  • 1 µg/ml ethidium bromide (store wrapped in foil at 4°C)
  • Plastic wrap
  • UV transilluminator
  • Additional reagents and equipment for gel photography (unit 2.5)
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.
   Thuring, R.W., Sanders, J.B., and Borst, P.A. 1975. Freeze‐squeeze method for recovering long DNA from agarose gels. Anal. Biochem. 66:213‐220.
   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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