Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/19 May 2015"

Revision as of 21:28, 19 May 2015

Starter culture failure
- Yesterday's attempt at creating a starter culture from the 5th plating of the old transformatants did you 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.
- Weighed wet pellet using analytical balance.

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+* Starter culture failure
+** [https://2015.igem.org/Team:UCLA/Notebook/Recombinant_Expression/18_May_2015 Yesterday's] attempt at creating a starter culture from the 5th plating of the old transformatants did you 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