Team:UCLA/Notebook/Honeybee Silk/20 July 2015

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The Cultures Didn't Grow

• The 14 cultures that I grew up yesterday, including the silk and pet vector in bl21 did not grow.
• We suspect that either the plates that we used to grow the cells on or the bl21 cells are bad.
• We are making new plates (Tristan) and I will run a test to see if a colony of the Bl21 cells grows in plain LB.

Redo of Transformation with pET+silk

We suspect there might be something wrong with the kanamycin plates we grew our colonies on, so today I will redo the transformation with the new plates.

1. Thaw cells (~80uL) on ice for 10 minutes.
2. Add 2uL of ligated product (diluted)
3. Place on ice for 5 minutes.
4. Rescue in 320uL of SOC, incubate and shake for 1 hour.
5. Warm kanamycin plates at 37C.
6. Spin down bacteria to pellet, discard supernatant.
7. Add 110 uL of SOC, take 10uL and dilute 1:10.
8. Plate 100uL of bacteria (1:1 and 1:10) on each plate.
9. Spread with beads, incubate at 37C.