Chromosomal DNA Isolation
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Chromosomal DNA Isolation
METHOD:
- Grow cells in 2-5 ml broth to late log phase.
- Pellet 1-2 ml cells in microfuge.
- Resuspend cells in 400 µl TES (50 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH7.5).
- Add: 17 µl 30% (w/v) sarkosyl (final concentration of 1%) and 5 µl 10 mg/ml proteinase K (final concentration 100 µg/ml).
- Incubate at 37ºC for 30-60 minutes, or until the solution clears.
- Add 400 µl 4 M NH4OAc and mix well.
- Extract 2X with 1:1 phenol:chloroform.
- Extract 1X with chloroform.
- Precipitate with an equal volume of isopropanol at RT for 10 minutes.
- Resuspend pellet in 400 µl 0.1 M NaOAc pH 6.0. Pellet may not resuspend completely. Reprecipitate DNA with 800 µl EtOH for 15 minutes at RT.
- Centrifuge at 11K for 15 minutes to pellet DNA.
- Rinse pellet in 1 ml 70% EtOH for 5 minutes.
- Dry pellet by placing open tube in 37C incubator or on bench top for 15-20 minutes.
- Resuspend DNA in 50 ul TE (10 mM Tris pH 8.0, 0.1 mM EDTA) overnight at 4C. May add 1 µl RNase to help DNA dissolve. Yields 25-50 ug of DNA; use 1-5 µl per digest.
TES:
5.0 ml 1 M Tris pH 7.5
0.2 ml 5 M NaCl
2.0 ml 0.5 M EDTA
93 ml sterile MQ water
