A. Reagents:

Lysis buffer:

0.45% Tween, 0.45% NP40, 2.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-Cl pH8.3, 100 µg/µl, Proteinase K

Tween20	225 µl
NP40	225 µl
1 M MgCl2	125 µl
1 M KCl	2.5ml
1 M Tris-Cl pH 8.3	0.5ml
20 µg/ml Prot K	250 µl*
dH2O	46.2 ml

* Add Proteinase K just before use, 5 µl stock per ml of lysis buffer used.

A. Protocol:

1. Wash cells with cold PBS.

2. Resuspend at 10,000 cells/µl in lysis buffer.

3. Incubate at 56°C for 1 hour.

4. Boil for 10 minutes.

5. Store lysate at -20°C

B. Reagents:

L6 buffer:

0.08 M GuSCN (LifeTechnologies GibcoBRL), 0.08 M Tris-Cl pH 6.4, 0.035 M EDTA, 2% (wt/vol) Triton X-100

B. Protocol^*:

1. Wash cells with cold PBS

2. Resuspend at 10⁶ cells/ml in L6 buffer.

3. Add an equal volume of Isopropanol.

4. Spin at 14000 rpm for 30 minutes at 4°C.

5. Discard supernatant.

6. Wash pellet twice with 75% Ethanol.

7. Airdry pellet.

8. Resuspend pellet in 100 µl dH₂O. Store at -20 °C.

* Adapted from Boom et al. J.Clin.Microbiol. 1991, 28:495-503.