SMALL SCALE PLASMID ISOLATION FOR XANTHOMONAS

SMALL SCALE PLASMID ISOLATION FOR XANTHOMONAS

CAPS BUFFER METHOD

  1. Spin down 1.5 ml of 2448 hr cells in microcentrifuge tubes at 7.5x (Beckman Microfuge 11) for 5 min.
  2. Pour off supernatant (make sure pellets are yellow for Xanthomonas). Carefully absorb remaining liquid with a Kimwipe; don't touch the pellet.
  3. Resuspend cells in 300 ul cold 50 mM CAPS, 3% SDS (pH 12.3 with 2 N NaOH). Keep SDS in suspension. Triturate pellet with transfer pipette until pellet is evenly suspended.
  4. Set tubes on ice for 10 min. To eliminate a majority of chromosomal DNA incubate tubes 1530 min in a 55 C water bath.
  5. Set tubes on ice for 5 min.
  6. Add 150 ul cold 5 M KAc (pH 4.8), invert tube and vortex gently for a few seconds.
  7. Put tubes on ice for 5 min, then microfuge at 13.5x for 15 min (top speed).
  8. Transfer supernatant to a fresh tube, measure volume (300400 ul).
  9. Add equal volume 'phenol/chloroform`. Vortex, centrifuge 13.5x for 5 min.
  10. Transfer aqueous phase to a fresh tube (take care not to collect any 'phenol/chloroform' or precipitate at the interface).
  11. Add two volumes 95% ethanol at room temperature. Vortex. Let stand at room temperature for 2 min.
  12. Set tubes in 20 C overnight or several hours.
  13. Centrifuge at 13.5x for 15 min. Pour off alcohol. Stand tubes inverted to drain liquid.
  14. Add 1 ml cold 70% ethanol if doing restriction digest, otherwise this step is not necessary. Vortex. Centrifuge at 13.5x for 5 min. Drain liquid.
  15. Dry pellet in vacuum desiccator (pull vacuum 1 min, close, leave for 510 min, bring to room pressure slowly).
  16. Dissolve pellet in 1535 ul autoclaved 10 mM Tris (pH 8), 1 mM EDTA, 20 ug/ml RNase (DNasefree). Store at 4 C, mix well before loading on a gel.

Notes:
Lysing buffer: dissolve CAPS and SDS in sterile distilled water. Adjust to pH 12.0 with 2 N NaOH (~5 ml NaOH per 100 ml lysing buffer). Do not stir solution while reading pH. Let solution stand 2 hrs. Recheck pH and adjust to pH 12.3 if necessary. Bring buffer up to volume. Lysing buffer can be used for up to one week. Do not use buffer if pH is 12.5 or over. Tris can be used in place of CAPS, but CAPS appears to work better.

5 M KAc: 60 ml 5 M potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml distilled water, autoclaved.

phenol/chloroform: 25:24:1 phenol, chloroform, isoamyl alcohol, respectively (phenol is first equilibrated with TE to pH 7.8).

TE with Rnase (20 ug/ml): 10 mM TRIS (pH 8.0), 1 mM EDTA, autoclaved, add DNasefree pancreatic RNase.