Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/19 May 2015"

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===Transformation===
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===Transformation with pET24a===
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#Dialyze 2 uL of pET24a expression vector on a dialysis membrane.
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#Thaw a 50uL aliquot of electro-competent E. coli cells on ice for 10 minutes. Chill transformation cuvettes on ice. Warm up SOC at 37C.
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#Add 1 uL of dialyzed pET24a vector to 50uL competent cell stock.
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#Add stock in between the plates of the electroporation cuvette.
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#Take out warmed SOC and prepare incubation tubes.
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#Place cuvette in pulse machine
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##Setting: 1.8 kV
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##Preload 950uL of SOC.
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#Press pulse and immediately add 950uL SOC to cuvette.
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#Pipet up and down two times, then add to incubation tube.
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#Incubate at 37C for one hour.
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##While waiting, warm up three kanamycin plates (1:1, 1:10, 1:100)
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#Make 1:10 and 1:100 dilutions with SOC.
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#Plate 100uL of each sample and spread with beads.
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#Incubate at 37C (incubated at 2:40 PM)

Revision as of 22:12, 22 May 2015

5/19

Cell Harvesting

  • Cells were grown up in 250 ml of LB medium and induced with IPTG. See 5/18 for details.
  • The Cells were split up into 5 pre weighed 50 ml falcon tubes, with 45 ml of cells in each tube.
    • I should have taken the OD 600 at this point, but I forgot to.
  • Mass Balanced the tubes and added a water tube, and spun down the cells at 5300 rpm at 4 C for 15 min. to pellet the cells.
  • Decanted the supernatant.
  • Weighed the pellets
    • In total, the wet weight of all the pellets across all 5 falcon tubes was 2.42 grams, approximately 0.5 grams per 45 ml of culture.
  • Froze down the pellets in the falcon tubes at -80C
  • Next up, will lyse the cells and collect protein with BugBuster


Transformation with pET24a

  1. Dialyze 2 uL of pET24a expression vector on a dialysis membrane.
  2. Thaw a 50uL aliquot of electro-competent E. coli cells on ice for 10 minutes. Chill transformation cuvettes on ice. Warm up SOC at 37C.
  3. Add 1 uL of dialyzed pET24a vector to 50uL competent cell stock.
  4. Add stock in between the plates of the electroporation cuvette.
  5. Take out warmed SOC and prepare incubation tubes.
  6. Place cuvette in pulse machine
    1. Setting: 1.8 kV
    2. Preload 950uL of SOC.
  7. Press pulse and immediately add 950uL SOC to cuvette.
  8. Pipet up and down two times, then add to incubation tube.
  9. Incubate at 37C for one hour.
    1. While waiting, warm up three kanamycin plates (1:1, 1:10, 1:100)
  10. Make 1:10 and 1:100 dilutions with SOC.
  11. Plate 100uL of each sample and spread with beads.
  12. Incubate at 37C (incubated at 2:40 PM)