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  Section: Biotechnology Methods » Molecular Biology
 
 
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Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Principle
A DNA restriction fragment is isolated by collection on DEAE cellulose paper during electrophoresis, then washed from the DEAE with a high salt buffer, cleaned, precipitated, and resuspended in a small volume. Recovery of 50%–90% of the bound DNA can be expected; however, fragments larger than 7 kb have lower yields. DNA prepared this way is suitable for subcloning.

A DNA fragment of a given size migrates at different rates through gels containing different concentrations of agarose. By using a gel at the appropriate gel concentration, it is possible to resolve well the DNA of interest. Use the following table as a guide for determining the agarose concentration to use.


Time Required
  • 3–4 hours on Day 1
  • 2–4 hours on Day 2
Special Materials
  • Schleicher & Schuell NA-45 DEAE membrane.
Procedure

Day 1
Isolating the fragment:
  1. Run the restriction digest and the appropriate size markers on a 1X Tris- Borate agarose gel with ethidium bromide. Be sure to leave at least 1–2 wells between samples. Run the gel until the DNA bands are well separated (visualize on the long-wavelength UV lightbox).
  2. Cut a slit just ahead of the band of interest using a sharp sterile razor blade or scalpel. Using blunt-edged forceps (such as Millipore forceps), carefully insert an NA-45 paper into the slit (prewet and cut NA-45 to the width of the band, see preparation of the NA-45 below).
  3. Place the gel in fresh 1X Tris-Borate buffer, and run the gel until the fragment has moved out of the gel and stopped by the NA-45. Monitor the progress of the band with the handheld long-wavelength UV light. Do not allow other bands of higher molecular weight to run onto the NA-45.
  4. Remove the NA-45 paper, rinse in NET buffer, and place in a labeled eppendorf tube. Add sufficient high-salt NET buffer to cover most of the membrane (typically 150–300 µL). Spin 5 seconds in a microcentrifuge to submerge the entire strip. Place at 65°C for 1 hour, mixing frequently, and respinning if the membrane rides up the side of the tube.
  5. Transfer the buffer (+DNA fragment) to a clean, labeled tube. Wash the membrane (in the original tube) with 50 mL high-salt NET buffer and add the wash to the DNA fragment tube.
Cleaning the DNA
  1. To remove ethidium bromide, extract twice with 3 volumes water-saturated n-butanol.
  2. Precipitate the DNA with 2.5 volumes of ethanol at –20°C for at least 1 hour (can sit overnight in the freezer).
Day 2
  1. Pellet the DNA (for 20 minutes at high speed in a microcentrifuge) and resuspend in 50 µL TE. Reprecipitate with sodium acetate to remove any residual NaCl. Add 5 mL 3M Na-acetate, and 120 µL ethanol, hold at –20°C for 2 hours or more, pellet as before, and resuspend in an appropriate amount of TE.

Solutions
  • Preparation of the DEAE cellulose membrane. Schleicher and Schuell NA-45 can be used as supplied by the manufacturer, with prewetting in sterile dH2O. However, the binding capacity of the membrane is increased with the following: a 10-minute soak in 10 mM EDTA pH 7.6, then 5 minutes in 0.5 N NaOH, followed by several rapid washes in sterile dH2O. Membranes can be stored for several weeks in sterile dH2O at 4°C.
  • NET buffer (500 mL). 25 mL 3 M NaCl, 150 mM NaCl, 100 mL 0.5 M EDTA, 100 mM EDTA,10 mL 1 M Tris pH 7.5, 20 mM Tris pH 7.5, 365 mL dH2O. Autoclave to sterilize.
  • High salt NET buffer (500 mL). 166.7 mL 3M NaCl, 1 M NaCl, 100 mL 0.5 M EDTA, 100 mM EDTA,10 mL 1M Tris pH 7.5, 20 mM Tris pH7.5, 223.3 mL dH2O. Autoclave to sterilize.

 
     
 
 
     




     
 
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