Bacterial Colony Fluorescence

Procedure

  1. Transform or electroporate library into an appropriate cell line.

  2. Perform dilution after transformation or electroporation to estimate total number of colony forming units (CFU).

  3. Dilute remainder of library to a final concentration of 20% glycerol for storage at -80 Celsius.

  4. Plate the remainder of the library, aiming for around 2000-3000 colonies per plate with arabinose present in the plate at a concentration of .02% mass fraction or an IPTG concentration of 1 mM (can be toxic at too high of concentrations).

  5. Pick clones for growth in 96-well Deep-Well plates.

  6. After sufficient time for growth, make 20% glycerol stocks, pool different clones, and subject to additional rounds of mutagenesis and selection.

Notes and Cautions

  • For pBAD, Omnimax, Electromax, and Top10 cells work best.

  • For pET vectors, Bl21 (DE3) pLysS is most appropriate.

  • If control of the induction time is necessary, plate the library on a nitrocellulose membrane, grow overnight, and transfer to arabinose or IPTG containing agar for the desired amount of time (e.g., for 3 hours at 37 Celsius).