Team:Newcastle/Project/Labwork/MoreProtocols

=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 37oC for 30 min.
 * Heat activated at 70oC for 30 min.
 * Germination in 10 mM L-alanine at 37oC.


 * 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. (NA0200S_NA0200). The list of plasmid kits can be accessed from 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 website (you will then need to open the Adobe file)

Lambda DNA/Hind III Digest
Lambda DNA/Hind III Digest