Extraction of genomic DNA
Published in Hughes and Welker 1988.
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[MATERIALS] - [INDEX]
Procedure
Note: Can use as little as one confluent plate (107) or 500 ml of 107 cells/ml.
Volumes in parentheses are suggested volumes for 108 cells. These should be adjusted if a
different number of cells is used.
- Collect cells.
- Wash once in KK2.
- Wash once in ice-cold 0.2% NaCl (= 0.034 M).
To decide volume to use: aim at 5-10 x107 cells/ml (1 ml).
Extract nuclei:
- Resuspend cells in ice-cold NP40-lysis buffer at 5-10 x107 cells/ml (1 ml).
- Vortex vigourously for 30 seconds.
Lyse nuclei:
- Add one volume of 2% SDS (1 ml).
- Incubate at 65°C for 15 minutes.
Purify DNA:
- Add one volume of TE, pH 9.5 (2 ml).
- Add proteinase K to 200 µg/ml.
- Incubate at 45-55°C for 2h to overnight.
- Cool briefly.
- Extract with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1).
- Repeat previous step until no more interphase is visible.
- Extract with chloroform:isoamyl alcohol.
- Precipitate DNA: add NaCl and ethanol, incubate at -20°C for >30 minutes.
- Resuspend in an appropriate volume of TE (100 µl).
- Quantify using OD260.
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Materials
- 1x KK2
- 0.2% NaCl
- NP40 lysis buffer
- 0.05M HEPES
- 0.05 M MgAc
- 10% sucrose
- 2% Nonidet P40 (NP40)
- 2% SDS
- TE, pH 9.5 (10 mM Tris, pH 9.5, 1 mM EDTA)
- Proteinase K (10-50 mg/ml)
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