Team:Newcastle/Project/Labwork/MoreProtocols

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(Other Lab Protocols)
(Recovery of cwlD spores)
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Sheffield graciously gave us some non-germinating spores. There are two methods which can be used to recover the cwlD spores, however, neither give complete restoration of germination.
Sheffield graciously gave us some non-germinating spores. There are two methods which can be used to recover the cwlD spores, however, neither give complete restoration of germination.
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=== Method A===
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==== Method A====
; Method A (fast method resulting in partial germination ~ approx. 0.1% recovery)
; Method A (fast method resulting in partial germination ~ approx. 0.1% recovery)
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: ''(Image missing)''
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=== Method B ===
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==== Method B ====
; Method B (slow method involving stripping of spore coat layers for improved germination ~ approx. 10% recovery)
; Method B (slow method involving stripping of spore coat layers for improved germination ~ approx. 10% recovery)

Revision as of 16:54, 12 August 2009


Other Lab Protocols

This page contains lab protocols which we have been given by other people. You may also be interested in Phil Aldridge's protocols.

This page contains the following protocols:


Recovery of cwlD spores

Sheffield graciously gave us some non-germinating spores. There are two methods which can be used to recover the cwlD spores, however, neither give complete restoration of germination.

Method A

Method A (fast method resulting in partial germination ~ approx. 0.1% recovery)

Sekiguchi, J., Akeo, K., Yamamoto, H., Khasanov, F., Alonso, J. & Kuroda, A. (1995). Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. J Bact 19; 5582-5589.

  • Spores suspended in lysozyme solution (200 g lysozyme per ml in10 mM potassium phosphate, 50 mM KCl, 1mM MgCl2).
  • Incubation at 37 oC for 30 min.
  • Heat activated at 70 oC for 30 min.
  • Germination in 10 mM L-alanine at 37 oC.
(Image missing)

Method B

Method B (slow method involving stripping of spore coat layers for improved germination ~ approx. 10% recovery)

Harwood, C. & Cutting, S. (1990). Modern microbiological methods: molecular biological methods for Bacillus. John Wiley and Sons Ltd., West Sussex, U.K.; 405-408.

Cleaning of spore surface by centrifugation (15,000 g, 20 min, 4 oC) using the following buffers:

  • TEP buffer , 0.5 M KCl, 1% glycerol
  • 1 M NaCl (twice)
  • TEP buffer, 0.1 % SDS
  • TEP buffer
  • 1M NaCl
  • dH2O, 0.01 % (w/v) Tween 80, 2 mM PMSF, 5 mM EDTA (twice)

(TEP buffer – 50 mM Tris-HCl pH7.4; 5 mM EDTA; 1 mM PMSF)

Removal of spore coat layers by alkali extraction:

  • Spores resuspended in 0.1 M NaOH
  • Incubate at 4 oC for 15 min (occasional vortexing)
  • Centrifuge (10,000 g, 10 min, 4 oC)
  • Pellet washed in dH2O (15,000 g, 10 min, 4 oC)
  • Heat activated at 70 oC for 30 min in Tris-HCl (pH 8.0 / 100 mM)
  • Germination in 10 mM L-alanine, 50 mM KCl.

Midiprep

We follow the protocol from GenElute for midipreps. (NA0200S_NA0200). The list of plasmid kits can be accessed from Gen Elute's plasmid kits page.




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