Team:UCLA/Notebook/Honeybee Silk/15 July 2015

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Inoculation

Sequencing results of pET24a+silk did not come back perfect. I picked 5 more colonies from the plate and am growing them up in 5mL LB broth and 5uL 1000X kanamycin. Incubated at 37C at 3:30PM.

Transformation with psb1c3+lac promoter+silk+spycatcher

1. Dialyze 2 uL of ligated vector+insert 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 2 uL of dialyzed vector+insert 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 chloramphenicol plates (1:1, 1:10)
10. Make a 1:10 dilution in SOC .
11. Plate 100uL of each sample and spread with beads.
12. Incubate at 37C (incubated at 4:30PM)