Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/12 July 2015"

(→‎Making competent Bl21(DE3) cells)
(→‎Cloning Honeybee Silk into pET vector)
 
Line 296: Line 296:
 
*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.
 
*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=
+
='''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)

Latest revision as of 21:28, 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.

Transformation with pET24a+AmelF3 (Honeybee silk)

  • Using C2987H cells


  1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
  2. Split into two aliquots of 25uL.
  3. Add 1uL of the 3:1 to one tube, and the 5:1 ligated product to the second aliquot.
  4. Place the mixture on ice for 30 minutes.
  5. Heat shock at exactly 42°C for exactly 30 seconds.
  6. Place on ice for 5 minutes.
  7. Pipette 850 µl of room temperature SOC into a culture tube.
  8. Pipette 100uL of RT SOC into the tubs, transfer each to a prepared culture tube (from last step)
  9. Place at 37°C for 60 minutes in the shaker incubator.
  10. Warm kanamycin plates to 37°C.
  11. Make 2 ten-fold dilutions in SOC.
  12. Plate 100uL of each dilution (1:1, 1:10, 1:100) and spread with beads.
  13. Incubate at 37C overnight (put in 1:00PM)