Materials

• Phenol
• Chloroform
• Absolute ethanol
• 70% ethanol
• TE buffer (10T/1E), pH 8.0
• 10% SDS
• 50 mg/ml lysozyme
• 5 M NaCl

Procedure

1. Macerate Cyanobacteria in TE Buffer (10T/1E)
2. Pellet the cells by centrifugation for 2 min at 10,000 rpm at room temperature
3. Remove supernatant. Add 500 µl TE buffer (10T/1E).
4. Add 1% SDS and 50 µl of 50 mg/ml lysozyme stock solution
5. Keep at 70°C for 15 min.
6. Add equal volume of Phenol : Chloroform
7. Centrifuge for 10 min at 10,000 rpm at room temperature
8. Take supernatant and extract it twice using 100% Chloroform extraction procedure.
9. Finally take supernatant and add 0.1 volume of 5M NaCl and 2 volumes of 100% ethanol and keep for precipitation at –20°C for 2 hrs
10. Centrifuge the tubes for 30 min at 15,000 rpm at 10°C
11. Wash the pellet twice by 70% ethanol by centrifuging it at 10,000 rpm for 10 minutes.
12. Dry the pellet and resuspend in TE buffer (10T/1E)