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Main purpose

• ON culture of Colony PCR products of HAO and AMO from 19-07-2013
• USER reaction of AMO and HAO with pZA21 (with native promoter).
• RFP amplification in pZA21 using primers without promoter

Who was in the lab

Henrike, Kristian, Julia

Procedure

USER reaction of AMO and HAO with pZA21 (with native promoter)

USER reaction was performed according to standard protocol (***).

USER mix:

• USER enzyme 1 uL
• NEB 4 buffer 0,5 uL
• 10x BSA 0,5 uL
• backbone pZA21 1uL

• Sample 1 - 3 uL of USER mix + 12 uL of AMO
• Sample 2 - Negative control, USER mix + 7 uL water

USER reaction was performed in PCR machine with programm: 37C for 40 min and then 25C for 30 min.

To 100uL of chemically competent E. coli cells 10 uL of USER mix after USER reaction was added.

Incubation of cells on ice for 30 minutes, heat shock at 42C for 90 sec. and 5 min on ice. Addition of SOC medium and 2 hours of incubation at 37C. Plating on plates with LB medium with Kanamycin, overnight incubation at 37C.