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From 2014.igem.org

Contents 1 Media 1.1 Agrobacterium 1.2 Marchantia 1.2.1 Marchantia plates 1.2.2 Selection plates 1.3 Transformation 1.3.1 Acetosyringone 1.3.2 Cocultivation 1.3.3 Agar Trap

Media

Agrobacterium

For plates, use standard LB + 1.5% agar + required antibiotics.

Marchantia

Marchantia plates

For marchantia, 1/2 Gamborg's B5 media + 1.2% agar is effective. Gamborg's B5 salts can be found readily from chemical suppliers.

Always use freshly autoclaved media, and work in a laminar flow hood, maintaining sterile conditions.

Fungal spores rapidly colonise the plates under conditions for Marchantia growth.

Always seal all plates using micropore tape.

Selection plates

If using the Marchantia polymorpha resistance cassette and Agrobacterium mediated transformation, use Marchantia plates with 10ug/ml hygromycin and 100ug/ml cefotaxime (to ensure that agrobacterium does not colonize the plate).

Note: selection plates do not contain a high enough concentration of hygromycin to preclude fungal colonisation. Work in sterile conditions.

Transformation

Acetosyringone

Use a fresh stock, with a concentration of 100mM, in DMSO. This will be used in transformation protocols as 1000x.

Acetosyringone cannot be autoclaved.

Cocultivation

For cocultivation you need 1/2 B5 (see above) + 5% sucrose (w/total v), with 100mM acetosyringone. Although sucrose is a sugar, its melting point is sufficiently high so that you can autoclave the 1/2B5 + 5% sucrose together, without needing to filter sterilise the sucrose.

Agar Trap

For AgarTrap, you should use MMA buffer. This is 10 mM MgCl2; 10 mM MES-NaOH, pH 5.7, with 150uM acetosyringone.

MES cannot be autoclaved, so will need to be filter sterilised prior to use.