Paul.xu at 16:55, 10 August 2016

2016-08-10T16:55:38Z

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== ATCC Protocol ==

T. thermophilus HB8 and its derivatives were grown at 70°C in TR medium as a rich medium  or at 55°C in synthetic medium as a nutritionally defined medium, and 1.5% gellan gum with 1.5 mM CaCl2 and 1.5 mM MgCl2 was added to TR medium for plates

 ATCC medium: 697 Thermus medium Yeast extract............................4.0 g Polypeptone Peptone (BD 211910)..........8.0 g NaCl.....................................2.0 g Agar....................................30.0 g Distilled water..........................1.0 L Adjust pH to 7.5.

== Japan Protocol ==

=== Liquid TR medium ===

T. thermophilus HB8 can be grown at 70°C in '''TR medium:''' 0.4% tryptone (Difco Laboratories, Detroit, MI, USA), 0.2% yeast extract (Oriental Yeast, Tokyo, Japan), and 0.1% NaCl, pH 7.5 (adjusted with NaOH).

T. thermophilus HB8 can be grown at 70°C in TR medium: 0.4% tryptone (Difco Laboratories, Detroit, MI, USA), 0.2% yeast extract (Oriental Yeast, Tokyo, Japan), and 0.1% NaCl, pH 7.5 (adjusted with NaOH).

The TR medium can be supplemented with 0.4 mM CaCl2 and 0.4 mM MgCl2 (TT medium) when used for the transformation experiments.

To make plates, 1.5% gellan gum (Wako, Osaka, Japan), 1.5 mM CaCl2, and 1.5 mM MgCl2 should be added to the TR medium (metals are necessary to solidify the gellan gum).

The minimal plates contained the following components (per l): 5 g sucrose, 0.75 g K2HPO4, 0.25 g KH2PO4, 2 g NaCl, 2.5 g (NH4)2SO4, 0.1 mg biotin, 1 mg thiamine, 125 mg MgCl2·6H2O, 25 mg CaCl2·2H2O, and 10 g gellan gum, pH 7.2 (adjusted with NaOH).

=== Liquid Medium 2 ===

Grow Cells at 75°C with vigorous shaking harvest by centrifugation before the end of the log phase of growth. 4 g yeast extract 6 g polypeptone

3 g NaCl per liter.

=== Transformation ===

 Add the following metal to TR medium

0.4 mM CaCl2

0.4 mM MgCl2.

=== Plate ===

Add

1.5% gellan gum (Wako, Osaka, Japan)

1.5 mM CaCl2, and 1.5 mM MgCl2 

to TR medium

=== Tryptophan auxotrophy ===

 To study the tryptophan auxotrophy, minimal plates should be supplemented with

11 μg/ml each of lysine, glycine, serine, methionine, and proline to assist cell growth.

The minimal plates contain the following components (per l):

 5 g sucrose, 0.75 g K2HPO4, 0.25 g KH2PO4, 2 g NaCl, 2.5 g (NH4)2SO4, 0.1 mg biotin, 1 mg thiamine, 125 mg MgCl2·6H2O, 25 mg CaCl2·2H2O, 10 g gellan gum, pH 7.2 (adjusted with NaOH).
[[Category:Buffer Recipe]]

Thermus thermophilus Culture - Revision history

Paul.xu at 16:55, 10 August 2016