DNA extraction from Gram Positive bacteria (Staphylococcus aureus): materials required and protocol

Materials required:

• Overnight culture of Staphylococcus aureus in LB
• TE buffer ( 10mM tris.Cl, 1mM EDTA, pH 8.0)
• 10% SDS
• 1% lysozyme
• 1:1 Phenol-Chloroform mixture
• Chloroform
• 5N NaCl
• 5M Ammoniun acetate
• Ice cold isopropanol
• 70% ethanol

Protocol of DNA extraction from gram Positive bacteria:

1. Take 1.5 ml of bacterial broth culture (overnight culture in LB) into a microfuge tube.
2. Centrifuge at 800rpm for 10 minutes at 4°C and discard the supernatant.
3. Suspend the pellet in 400µl TE buffer. Mix well by vortexing.
4. Add 20µl of 1% lysozyme, mix well and incubate at 37° for 10 minutes in water bath.
5. Add 30µl of 10% SDS and mix it.
6. Incubate the tube at 37°C for 30 minutes in water bath.
7. Shear the cell suspension 3-5 times with the help of 26G needle.
8. Add 500µlof 1:1 phenol-chloroform mixture.
9. Centrifuge at 13000rpm for 2 minutes at 4°
10. Transfer the supernatant into another microfuge tube.
11. Add 500µl of chloroform and centrifuge at 13000rpm for 2 minutes at 4°
12. Transfer the supernatant into another microfuge tube.
13. Add 25µl of 5N NaCl and centrifuge at 13000rpm for 10 minutes at 4°
14. Discard the supernatant and suspend the pellet in 100µl TE buffer.
15. Incubate the tube at 37°C for 30 minutes.
16. Add 50 µl of 5M ammonium acetate.
17. Add 250 µl of cold isopropanol and incubate at room temperature for 5 minutes.
18. Centrifuge at 13000rpm for 5 minutes at 4°
19. Discard the supernatant and wash the pellet with 100 µl of 70% ethanol.
20. Pour off the ethanol and invert the tube on a clean absorbent paper to drain.
21. Allow the pellet to air dry for 5-10 minutes.
22. Suspend the pellet in 100µl TE buffer.
23. Store at -20°

DNA extraction from Gram Positive bacteria (Staphylococcus aureus): materials required and protocol