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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 cw1D spores
• Conducting a Midi prep
• Inducing competence in JM109 E.coli cells

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 in 10 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.

In addition to the protocol, it is important to take note of the following points

• Typically, 10ul of cwlD spores are added to the lysozyme and buffer solution.
• After the addition of L-alanine to the solution (which would initiate germination), the solution should be left in the incubator for 10 minutes.
• After 10 minutes, the eppendorf tube containing the solution should be spinned down for approximately a minute.
  • Note: Another eppendorf tube containing water should be placed on the opposite site to balance out the weight.
• After spinning down the solution, the supernatant should be removed and the spores should be resuspended in 1000ul of LB.

(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. ([http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/na0200bul.Par.0001.File.tmp/na0200bul.pdf NA0200S_NA0200]). The list of plasmid kits can be accessed from [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/hp-plasmid-kits.html Gen Elute's plasmid kits page].

Inducing competence in JM109 E.coli cells

For these E.coli cells a protocol devised by Promega (the company from which the competence agents were purchased) was used - see [http://www.promega.com/tbs/tb095/tb095.html website] (you will then need to open the Adobe file)

Lambda DNA/Hind III Digest

[http://www.imbb.forth.gr/groups/minotech-new/pdf/lambda_DNA_HindIII_digest.pdf Lambda DNA/Hind III Digest]