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Summary: 71
Yeast Genomic DNA Preparation from Spheroplasts
1. Resuspend with 50 mM Tris, 25 mM EDTA (pH 8) in 10X the spheroplast pellet volume.
2. Immediately add 1/10 volume of 10% SDS. Mix.
3. Incubate 30 minutes at 65°C. Place tubes on ice 5 minutes.
4. Measure volume. Add 1/3 volume of 3M potassium acetate, pH 5. Mix gently.
5. Hold tubes on ice for 30-60 minutes. Transfer to two 30 ml Corex centrifuge tubes.
6. Centrifuge 30 minutes in SS-34 rotor at 10,000 rpm at 4°C.
7. Transfer supernatant to fresh 30 ml Corex tubes. Add equal volume of phenol:CHCl3. Mix.
Centrifuge 10 minutes in SS-34 rotor at 10,000 rpm at 4°C.
8. Transfer supernatant to fresh 30 ml Corex tubes. Measure volume. Add 2 volumes of 100%
ethanol. Mix gently. DNA may precipitate immediately. Precipitate on ice for 1 hour.
9. Centrifuge 10 minutes in SS-34 rotor at 10,000 rpm at 4°C. Discard supernatant.
10. Wash with 70% ethanol. Gently break up pellets. Wash for at least 5 minutes at 25°C.
11. Centrifuge 10 minutes in SS-34 rotor at 10,000 rpm at 4°C. Discard supernatant.
12. Drain and dry pellets on bench. Dissolve each pellet completely in 2 mls TE (pH 8).
13. Add 20 µl 10 mg/ml RNase A (heat treated; DNase-free) to each tube. Incubate for 30 minutes
at 37°C. The digestion may be stored overnight at 4°C at this point.
14. Centrifuge 5 minutes in SS-34 rotor at 10,000 rpm at 4°C. Transfer supernatant to a fresh 30 ml
Corex tubes.
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