Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/19 May 2015"
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* Starter culture failure | * 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 | + | ** [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 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. | *** 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. | **** Incubated plates at 37 degrees shaking at 1415. Will check OD of starter cultures at 1900. | ||
Revision as of 21:29, 19 May 2015
- Starter culture failure
- 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.
- 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.
- 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.
- 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).
- Inoculated 950 mL of LB supplemented with 1mL of 100x Carbenicillin (final concentration 100ug/uL) with 10mL starter culture grown overnight.
- 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.