Team:UCLA/Notebook/Honeybee Silk/12 July 2015
Making competent Bl21(DE3) cells
- The starter culture from 7/11 overgrew to around an OD of 3 (want OD around 0.5) so I am re-picking colonies to make a new starter culture in 7 ml of LB broth. Put in 37 C incubator t 9:40 AM
- Using [http://www.zymoresearch.com/downloads/dl/file/id/166/t3001i.pdf this] protocol.
- At 1 pm (3 h 20 m) the OD was up to 0.52, (shooting for between 0.4 and 0.6)
- Added 0.5 ml to 50 ml of zymo broth. Left at RT for an hour and placed into 21 C incubator starting at 2 pm 7/12.
- The incubator started at around 27 C at 2 pm and is taking a while to ramp down.
- At 5:50 Pm the OD is 0.087, which seems rather high for only 4 hours. The protocol says 10 to 36 hours.
- At 7:50 pm (6 hour incubation time) The OD is 0.45 and I stopped incubating and began the protocol for making cells.
- After completion of the protocol, I aliquoted the cells (100 ul) into 50 pcr tubes in the -80C, second row fourth unit from the bottom.
Transformation with pET24a+AmelF3 (Honeybee silk)
- Using C2987H cells
- Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
- Split into two aliquots of 25uL.
- Add 1uL of the 3:1 to one tube, and the 5:1 ligated product to the second aliquot.
- Place the mixture on ice for 30 minutes.
- Heat shock at exactly 42°C for exactly 30 seconds.
- Place on ice for 5 minutes.
- Pipette 850 µl of room temperature SOC into a culture tube.
- Pipette 100uL of RT SOC into the tubs, transfer each to a prepared culture tube (from last step)
- Place at 37°C for 60 minutes in the shaker incubator.
- Warm kanamycin plates to 37°C.
- Make 2 ten-fold dilutions in SOC.
- Plate 100uL of each dilution (1:1, 1:10, 1:100) and spread with beads.
- Incubate at 37C overnight (put in 1:00PM)