Yesterday's attempt at creating a starter culture from the 5th plating of the old transformatants did NOT grow. We hit an OD600 of 0.004, basically negligible growth.
Created new 11mL LB supplemented with 11uL 1000x Ampicillin (final 1x concentration 50ug/uL) starter cultures with colonies picked from 1/21 platings -- Plates 1,2,4,5.
Incubated plates at 37 degrees shaking at 1415. Will check OD of starter cultures at 1900.
1L Expression of Tamura construct
Inoculated 950 mL of LB supplemented with 1mL of 100x Carbenicillin (final concentration 100ug/uL) with 10mL starter culture grown overnight.
Shook cells in 37 degrees Celsius until an OD600 of 0.6 is reached.
Induced protein expression using 5mL of 100mM IPTG stock (final concentration 0.5mM).
Gene expression monitoring
Removed 1mL of culture before adding IPTG and at 1-hour intervals after addition of IPTG, until after 5 hours after gene expression induction.
Spun down cells for 15 minutes at 5,300 ref at 4 degrees Celsius, and decanted supernatant.