Revision as of 06:05, 13 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.

@@ Line 293: / Line 293: @@
 **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.
 =Cloning Honeybee Silk into pET vector=