Back to Timeline . Recipes for Mixes
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 in a 37 degree shaker for 0-60 minutes before plating.
11) For source plate DNA, plate 100 microliters.
Enrichment cultures
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.
For both: culture initially for 3 days, then reculture for 7 days. Then test for DNA and continue cultures.
Qiagen Miniprep kit: www.qiagen.com/hb/qiaprepminiprep
Mobio PowerMax Soil kit: http://www.mobio.com/images/custom/file/protocol/12988-10.pdf
Pulse Gel Field Electrophoresis:
PFGE separation of 0.5 µg of Lambda Mono Cut Mix, 0.1% agarose gel, 0.5X TBE
Parameters: 6 V/cm, 15°C for 20 hours.
Switch times ramped from 0.5-1.5 seconds.
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