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.

Department of Microbiology
School of Medicine
University of Washington
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