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From 2008.igem.org

We searched the registry to try and find a broad host range, Gram positive plasmid (pTG 262), but discovered that although it had a BioBrick number (BBa_I742103) it hadn't made it into the 2008 parts registry. Oh well!

Instead, we (Dave and Lisa) chemically transformed competant E. coli using our PAC and pUC-EUY plasmids, in order to grow up some more copies of our carotenoid genes.

I poured four plates of LB medium, 2 with chloramphenical, two with kanamycin (ampicilin analogue)

This was our protocol:

-Prep ice bucket, pre-warm 150 uL of sterile LB medium to 37 *C -Add 1/2 to all (or at least 1 ug of DNA) of ligation mix to competant E. coli cells (previously frozen at -80 *C) -Ice cells + ligation mix for 30 minutes, flicking every ten minutes to mix -Heat shock at 42 *C for exactly 1 minute -Quickly add the prewarmed LB to the tube -Ice two minutes -Incubate at 37* on shake table for 30 minutes -Plate mix on selective media, culture overnight at 37*C

Errata:

-We left the transformation mixture on ice longer than 30 minutes, as the heating block was too hot initially (60 *C instead of 42) -We iced for four minutes, rather than two, as I misread a handwritten '2' as a '7'.

Minor ramblings 15:13, 24 July 2008 (UTC)

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