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Home :: Technical Resources :: Genomic Purification
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Genomic Purification
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Once DNA has been extracted from tissue, it is necessary to remove proteins and other substances. If the DNA samples have not been cleaned sufficiently or have been degraded even slightly, results of PCR may not prove satisfactory. If there is a question as to purity, the following procedure may improve PCR results:
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- Add 25% volume of phenol:chloroform:isoamy alcohol (25:24:1) to the sample, mix well.
- Centrifuge at 5000 X g for 10 minutes.
- Draw off phenol:chloroform:isoamy alcohol (organic bottom layer).
- Add 25% volume of chloroform, mix well.
- Centrifuge at 5000 X g for 10 minutes.
- Draw off chloroform (organic bottom layer).
- Centrifuge again at 5000 X g for 10 minutes.
- Draw off remaining chloroform.
- Add 10% volume of 3 M sodium acetate and 2 volumes of 95% ethanol, mix well.
- Place at -20°C for a minimum of 3 hours, preferably overnight.
- Centrifuge at 5000 X g for 45 minutes.
- Draw off or decant ethanol, being careful not to disturb the pellet.
- Add 80% ethanol at 50% of the original sample volume.
- Centrifuge at 5000 X g for 45 minutes.
- Draw off or decant ethanol, being careful not to disturb the pellet.
- Dry pellets, then add 1 mM Tris HCl pH 8.0, 0.1 mM EDTA, 0.01% Tween-20 to a volume allowing for concentration determination by spectrophotometric or fluorometric measurement.
- Dilute DNA with 1 mM Tris HCl pH 8.0, 0.1 mM EDTA, 0.01% Tween-20 to desired concentration.
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