|  |  | | | | | Contributor: | The Laboratory of Donald Rio at the University of California, Berkeley
| | | A. Restriction Digest
1. Cut 10 to 15 μg of CsCl-purified plasmid DNA containing the fragment of interest with 2-fold excess of Restriction Enzyme for 2 hours (see Hint #2 and #3).
2. Add an equal volume of Phenol:Chloroform, mix well by inversion, and centrifuge in a microcentrifuge at full speed for 3 min to separate the phases. Save the aqueous phase (upper layer).
3. To the aqueous phase, add an equal volume of Chloroform, mix well by inversion, centrifuge in a microcentrifuge at full speed for 3 min to separate the phases. Save the aqueous phase (upper layer).
4. To precipitate the DNA, add 0.1 volumes of 3 M Ammonium Acetate and 2.5 volumes of 100% Ethanol to the aqueous phase and mix well by inversion.
5. Centrifuge the tube in a microcentrifuge at full speed for 5 min to pellet the DNA and discard the supernatant.
6. Wash the pellet with 70% Ethanol, mix well by inversion, and centrifuge in a microcentrifuge at full speed for 5 min to pellet the DNA. Discard the supernatant.
7. Dry the DNA pellet by vacuum centrifugation.
8. Resuspend the pellet in 180 μl of ddH2O.
B. Calf Intestine Phosphatase Treatment
1. Add 20 μl of 10X CIP Buffer.
2. Add diluted CIP stock to the DNA such that the final concentration is approximately 0.2 Units CIP per μg DNA in a final volume of 200 μl (see Hint #4).
3. Incubate at 37°C for 1 hour (see Hint #5).
4. Add an equal volume of Phenol:Chloroform, mix well by inversion, centrifuge in a microcentrifuge at full speed for 3 min to separate phases and save the aqueous phase (upper layer).
5. To the aqueous phase, add an equal volume of Chloroform, mix well by inversion, centrifuge in a microcentrifuge at full speed for 3 min to separate the phases. Save the aqueous phase (upper layer).
6. To precipitate the DNA, add 0.1 volumes of 3 M Ammonium Acetate and 2.5 volumes of 100% Ethanol to the aqueous phase and mix well by inversion.
7. Centrifuge in a microcentrifuge at full speed for 5 min to pellet the DNA. Discard the supernatant.
8. Wash the pellet with 70% Ethanol, mix well by inversion, and centrifuge in a microcentrifuge at full speed for 5 min to pellet the DNA. Discard the supernatant.
9. Dry the pellet by vacuum centrifugation.
10. Resuspend the dried DNA in ddH2O to a final concentration of approximately 500 μg/ml.
11. Store at -20°C.
C. Kinasing Reaction
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