Extraction of genomic DNA

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.

  1. Collect cells.

  2. Wash once in KK2.

  3. 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:

  1. Resuspend cells in ice-cold NP40-lysis buffer at 5-10 x107 cells/ml (1 ml).

  2. Vortex vigourously for 30 seconds.

Lyse nuclei:

  1. Add one volume of 2% SDS (1 ml).

  2. Incubate at 65°C for 15 minutes.

Purify DNA:

  1. Add one volume of TE, pH 9.5 (2 ml).

  2. Add proteinase K to 200 µg/ml.

  3. Incubate at 45-55°C for 2h to overnight.

  4. Cool briefly.

  5. Extract with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1).

  6. Repeat previous step until no more interphase is visible.

  7. Extract with chloroform:isoamyl alcohol.

  8. Precipitate DNA: add NaCl and ethanol, incubate at -20°C for >30 minutes.

  9. Resuspend in an appropriate volume of TE (100 µl).

  10. 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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