Latest revision as of 20:53, 24 August 2015

Redo of Expressing Honeybee Protein

Following a similar protocol to the one performed on 5/18.
Growing up cells in 100ml of LB.
- We are doing a smaller volume because we are running low on bugbuster lysis reagents, and we want to be able to do at least one more reaction after this one.
Instead of making a starter culture, we are simply inoculating a colony into a 1L flask.
I started incubating the culture at 12:35 pm 7/27, but I forgot to add the Kanamycin antibiotic until 1 pm. (100 ul of 1000x).
Here are the steps for today's growth protocol.
1. Inoculate one colony in 100 ml of LB in an autoclaved flask.
2. Add appropriate antibiotics (kan 100 ul)
3. Grow and shake at 37 until OD reaches between 0.4 and 0.6.
  - The colony was inoculated at 12:20 pm, At 4 pm , the OD was 0.064, at 5:05 pm OD was 0.358, At 5:17 the OD was 0.502, when we induced with the IPTG (5:25 pm) IPTG was added to a concentration of 0.5mM

1. Continue to incubate at 37 C and shake overnight.
2. In the morning, spin down the cells, decant the supernatant, and freeze the pellet at -80 for storage.

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-='''Transformation of psb1c3+T7 promoter+silk into BL21(DE3)'''=
-#Thaw cells (~80uL) on ice for 10 minutes.
-#Add 1uL of ligated product.
-#Place on ice for 5 minutes.
-#Rescue in 320uL of SOC, incubate and shake for 1 hour.
-#Warm chloramphenicol plates at 37C.
-#Spin down bacteria to pellet, discard supernatant.
-#Add 111 uL of SOC, take 11uL and dilute 1:10 (11uL into 99uL).
-#Plate 100uL of bacteria (1:1 and 1:10) on each plate.
-#Spread with beads, incubate at 37C.
 ='''Redo of Expressing Honeybee Protein'''=
 *Following a similar protocol to the one performed on [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/18_May_2015 5/18.]