Team:Caltech/Protocols

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Caltech iGEM 2011

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Transforming DNA from Distribution Plates:
1) Thaw competent cells on ice.
2) Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.
3) Transfer into storage tube.
4) Pipette 1-2 microliters of the DNA into the competent cell tubes.
7) Stir with pipette tip, gently flick tube.
8) Leave on ice for 30 minutes.
9) Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.
10) Pipette 500 micro Liters of S.O.C. (LB + glucose) into 14ml culture tubes and transfer the competent cells into these tubes and incubate for 0-60 minutes before plating in a 37 degree shaker.
11). For source plate DNA, plate 100 microliters.

Enrichment cultures

For BPA (since soluble)

1) Set up 16 tubes: 8 tubes with vitamin media vs. 8 tubes with media (no vitamin), 4 tubes for each of the four locations.
2) Place 8 test tubes in 30°C shaker and 8 test tubes in room temperature shaker.

For 17α-estradiol, DDT, and nonylphenol (since non-soluble)

1) Set up two flasks: one with vitamin media, one without vitamin.
2) Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask.