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Method: Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
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.
|% agarose in the gel ||Efficient range of separation of linear DNA molecules (kb)|
|0.3 ||60 - 5|
|0.6 ||20 - 1|
|0.7 ||10 - 0.8|
|0.9 ||7 - 0.5|
|1.2 ||6 - 0.4|
|1.5 ||4 - 0.2|
|2.0 ||3 - 0.1|
- 3-4 hours on Day 1
- 2-4 hours on Day 2
Schleicher & Schuell NA-45 DEAE membrane.
Isolating the fragment:
- 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.
- 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).
- 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 hand-held long wavelength UV light. Do not allow other bands of higher molecular weight to run onto the NA-45.
- 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 Ál). Spin 5 seconds in a microcentrifuge to submerge the entire strip. Place at 65 degrees C for 1 hour, mixing frequently, and respinning if the membrane rides up the side of the tube.
- Transfer the buffer (+ DNA fragment) to a clean labeled tube. Wash the membrane (in the original tube) with 50 Ál high salt NET buffer and add the wash to the DNA fragment tube.
Cleaning the DNA:
- To remove ethidium bromide, extract twice with 3 volumes water-saturated n-butanol.
- Precipitate the DNA with 2.5 volumes of ethanol at -20 degrees C for at least 1 hour (can sit overnight in the freezer).
- Pellet the DNA (for 20 minutes at high speed in a microcentrifuge) and resuspend in 50 Ál TE. Reprecipitate with sodium acetate to remove any residual NaCl: Add 5 Ál 3M Na-acetate, and 120 Ál ethanol, hold at -20 degrees C for 2 hours or more, pellet as before and resuspend in an appropriate amount of TE.
- 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: 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 degrees 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 dH20 Autoclave to sterlize
- 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
Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp 6.24-6.27.