Caltech/Protocols/Two Layer


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iGEM 2007

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This requires only a simple modification of the standard titering protocol.

1. Perform the standard titering protocol using an amber suppressor strain (e.g. E. coli LE392) as the host cell to be infected. Make sure to simultaneously prepare a culture of non-amber-suppressor cells (e. g. E. coli MG1655) grown to a similar OD as the amber suppressors.

2. After the agar layer with LE392 is plated and allowed to solidify for a few minutes at room temperature, add 0.1 mL of non-amber suppressor (e.g. E. coli MG1655), also grown to OD ~ 0.1, to 3mL of the same cooled top agar, and pour this layer over the suppressing layer. Allow this second layer to cool, and then incubate.