Preparation of mycoplasma chromosomal DNA in TE

1. Spin 50 ml freshly grown cells.

2.Wash wash pellet twice with PBS.

3. Resuspend the pellet in 1ml TNE buffer ( same buffer as above. use 2 tubes, 500 ul each).

4. Add 5 ul proteinase K and incubate for 3-5 hours at 37 C. (During this period, more proteinase K can be added).

5. Add 1/5 (100 ul) volume of lysis buffer and sharply invert 1-2 times until the solution becomes clear. Then add 100 ul more lysis buffer.

6. Incubate at 37 C overnight.

7. Add equal volume (700ul) of equilibrated phenol and mix for 5 minutes and spin 15 minutes. Repeat this step until no proteins are present in the interface.

8. Centrifuge and resuspend pellet in TE buffer.

9. Wind out the DNA with pipitte.

10. Vacuum dry and suspend DNA in water or TE. Place at 4 C for several days to equilibrate before measuring DNA concentration, etc.

Lysis buffer: 1% NP-40, 1% Tween-20 and 1% deoxycholate.