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8. Seal one end of treated dialysis tubing with a dialysis clip. *After electroelution, it is recommended that the DNA is further purified with a phenol/chloroform extraction followed by ethanol precipitation. Oligosaccharides and other contaminants (found in low-grade agarose) can copurify with the DNA. Phenol extractions will remove any oligosaccharides, avoiding their coprecipitation during ethanol precipitations. 9. Fill the bag to the top with 1X TAE buffer. 10. Transfer the slice of agarose into the bag with the spatula.
When the pipette is removed from the gel a small plug of agarose will be contained in the tip. 1 ng/µl. 7. Add a portion of the DNA into the amplification reaction mixture (do not exceed 1 ng DNA/reaction). 4. Remove the plug of agarose. 5. Add a portion of the plug of agarose to the amplification reaction. The plug does not require melting; it will melt during the first denaturing step. Amplification Reaction Mixture If the reaction buffer does not contain magnesium ion, add sufficient amount for your template/primer.
Three sheets of soaked Whatman 3MM chromatography paper. 3B. A piece of positively charged nylon membrane. 3C. Agarose gel (underside of the gel in contact with the membrane). 3D. Three sheets of soaked Whatman 3MM chromatography paper. 4. Allow excess buffer to drain off the sandwich. 5. Load the sandwich into the semi-dry blotting apparatus. 6. Turn on the power and transfer by running at: 6A. 6 volts for 15 minutes 6B. 12 volts for 30 minutes 7. Remove the membrane from the blotting apparatus.
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