DNA isolation (glass bead) from Wangiella dermatitidis
P. McIntosh

(Adapted from Current Protocols in Molecular Biology, Volume 2, Section IV, Unit 13.11).

Materials
Protocol
Results
Tips

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25:25 buffered and purified phenol:chloroform solution.

Breaking buffer (add in order listed below)

     2% (v/v) Triton X-100

     1% (w/v) SDS

     100 mM NaCl

     10 mM Tris-Cl, pH 8.0

      1 mM EDTA, pH 8.0

RNAse A

Lead glass beads (400-520 µm diameter). Aliquoted into vials (0.3 g per vial)

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Collect 10 ml of a lag phase culture of W. dermatitidis grown in YPD broth.

2. Centrifuge cells for at 1200-1800 g for five minutes.

3. Remove supernatent and add 0.5 ml deionized (DI) H₂O.

4. Centrifuge cells for at 1200-1800 g for 5 minutes, then remove all remaining supernatent.

5. Resuspend cells in 200 µl breaking buffer and transfer to 1.5 ml Ependorf microtube.

6. Add 200 µl phenol:chloroform and 0.3 g glass beads and close cap tightly.

7. Place in microtube rack and put rack in a plastic bag.

8. Vortex at full speed for 6 1/2 minutes (VWR multitube vortexer).

9. Add 200 µl TE buffer and flick tube to mix.

10. Microcentrifuge at full speed for 5 minutes.

11. Collect top layer into clean microtube.

12. Add 1 ml 100% ethanol and invert to mix. Nucleic acids will coagulate.

13. Microcentrifuge at full speed for 3 minutes.

14. Suspend pellet in 200 µl TE buffer, add 1.5 µl RNAse A. Incubate at 37°C for 1 hour.

15. Add 5 µl 5 M potassium acetate, 0.5 ml 100% ethanol and invert to mix. DNA will coagulate.

16. Microcentrifuge at full speed for 3 minutes.

17. Remove supernatent and add 200 µl ice cold 70% ethanol.

18. Microcentrifuge at full speed for 3 minutes.

19. Remove supernatent and dry pellet in 37°C water bath or dessicator for 15 minutes (correct time depends on amount of residual liquid).

20. Resuspend pellet in 50 µl DI water in an ice bath with occasional flicking. After full suspension, DNA is ready for use or storage at -20°C.

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a) If coagulation is poor during step 12, try making fresh phenol:chloroform solution.

b) If aqueous layer is indistinct or unstable after step 10, add 1 part isoamyl alcohol to your phenol:chloroform solution.

c) 10 ml of a 3-4 day old culture will yield enough DNA to perform 8-12 Southern blots.

d) The plastic bag used in step 7 provides protection in case of leakage of the tubes during vortexing.

e) Breaking buffer can be stored at room temperature for up to a year.

f) Use of phenol:chloroform should be done only in a fume hood.

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This page updated on:
Thursday, June 19, 2003 04:33:58 PM