Preparation of Mycoplasma chromosomal DNA in low melting agarose

1. Spin 1.5ml fresh cells for 3 minutes.

2.Wash the pellet once with TNE buffer.

3. Suspend the pellet well in 20 ul TNE buffer.

4. Add 2 ul proteinase K ( stock 10 ug/ul in water, -20 C).

5. Add 20 ul low melting agarose (1.6% in TNE buffer. prewarmed).

6. Place on ice for 3-5 minutes and transfer the solid plug into polystyrene tube containing 0.5 ml 2% tween-20 in TE

7. Incubate at 37 C overnight.

8. Dialyse the plug in TE at 4 C for at least four hours. Change buffer every 1 hour.

TNE: 10 mM TrisHCl (pH 8.0); 150 mM NaCL; 1 mM EDTA