Protocol: Chromosomal DNA Isolation from Bacteria

1. Spin down 50-100 ml well-grown bacteria, 3600 rpm,15min.

2. Resuspend bacteria with 20 ml Buffer S, immediately add 100 µl ProteinaseK (10 mg/ml). Vortex to make sure no chunks.

3. Add 2 ml of 20% SDS, mix gently by inverting.

4. Incubate the mixture at 65 ^oC for 1 hr with inverting every 15 min.

5. Add 10 ml of phenol and 10 ml of chloroform, mix thoroughly by inverting for 5 min, spin at 3600 rpm for 20 min.

6. Transfer supernatant to a new tube, add 0.6 volume of isopropanol. Mix gently by inverting. You will see cotton-like genomic DNA.

7. Hook out the cotton-like DNA to a 1.5 ml tube; wash with cold 70% ethanol.

8. Dry DNA at RT, dissolve DNA in 500 µl H₂O (50 ^oC or 4 ^oC overnight).

9. Add 5 µl DNase-free RNase A (20 mg/ml stock) to the DNA. Incubate at 37 ^oC for 30 min.

10. Add 500 µl phenol, mix, spin at 3600 rpm for 20 min. Repeat phenol extraction once if necessary.

11. Transfer supernatant to new tube, add 1/10 volume of 3M sodium acetate and 2 volume of ethanol, mix gently. You will see cotton-like DNA.

12. Hook out the cotton –like DNA, wash with cold 70% ethanol.

13. Dissolve DNA in 400 µl sterile H₂O.

Yield: ~350 µg of genomic DNA from 100 ml of Desulfovibri vulgaris Hildenborough culture.

Solutions

Buffer S

CTAB/NaCl solution: 10% CTAB in 0.7 M NaCl

Dissolve 4.1 g NaCl in 80 ml distilled H₂O and slowly add 10g CTAB (Sigma M-7635) while heating and stirring. Adjust final volume to 100 ml.

CTAB: Hexadecyltrimethylammonium bromide; Cetyltrimethylammonium bromide; Cetrimonium Bromide; Cetab; Centimide

Proteinase K (10 mg/ml)

Buffer

Dissolve 100 mg Proteinase K in 10 ml of Buffer. Aliquot and store at -20oC.

3M sodium acetate (pH5.2)

Dissolve 204.15 g of sodium acetate·3H₂O in 300 ml of H₂O. Adjust pH to 5.2 with glacial acetic acid.

Adjust final volume to 500 ml.

Sterilize by autoclaving.

BIOMAG