Protocol 2.2 through 2.4

Protocol 2.2 through 2.4 - Protocol 2.2 Conjugation of...

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Protocol 2.2 – Conjugation of pDN19-wspR plasmid and Identification of Auxotrophs Protocol 2.2.1: Introducing plasmid encoding wspR into the bacterium Protocol 2.2.1 begins similarly to protocol 2.1. Use cotton swabs to collect bacteria ( P. aeruginosa from protocol 2.1, and E. coli DH5a pDN19 -wspR ) from plate and add to the appropriate tube with 1 mL of Lysogeny broth. Combine 100 microliters of the aeruginosa tube with 100 microliters of the E. coli tube and pipet up and down to mix. Spot 50 µL of the third tube (the “mating mixture” on an LB plate. Incubate the tube at 37°C for 90 minutes. Prepare 10 -2 , 10 -3 , and 10 -4 dilutions and plate 100 µL of each onto 3 LB/Gn/Irg plate, as well as 100 µL of an undiluted control. After completing protocol 2.2.2, incubate for 24 hours at 37°C. Protocol 2.2.2: Identifying Auxotrophic Mutants With Replication Plates Before putting away the plates from 2.2.1 (or in case a change by the TA mandates it, the plates from Tuesday’s 2.1), put two folded Kimwipes on top of the replica plating block
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This lab report was uploaded on 04/08/2008 for the course BSCI 412 taught by Professor Lee during the Spring '08 term at Maryland.

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Protocol 2.2 through 2.4 - Protocol 2.2 Conjugation of...

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