Team:Carnegie Mellon/Protocols

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His-Tag Soluble Protein Extraction
  1. Centrifuge 1.5 ml of culture for 1min full speed at 15000 rpm.
  2. Remove supernatant.
  3. Add 250 µl of extraction buffer (1% octyl-beta-thioglucoside in 10mM Tris-Cl, pH7.5).
  4. Incubate at room temperature for 10 min.
Wash Beads
  1. Remove 50 µl of Ni-NTA in 10 mM Trischloride bead solution and place in new tube for each purification.
  2. Add 1 mL buffer (see table).
  3. Centrifuge at 800 rpm for 30 seconds.
  4. Remove supernatant.
  5. Repeat 3 times and resuspend in 1 mL of Bead Wash Solution.
Estrogen Sensor
Estrogen Sensor Protocol
MiniPrep Protocol
Minipreps of MACH Cells Expressing Flourescence
Purpose: To isolate plasmid DNA from MACH cells.

Procedure:

  1. Set up overnight cultures for miniprep.
  2. Make the following reaction recipe:
    • 5 mL LB
    • 5 µL Chlorophenical
    • 1 µL overnight colony
  3. Incubate in 37°C for 16-18 hours.
  4. Follow the Life Technologies Miniprep Kit Protocol.
Restriction Enzyme Digest Protocol
Restriction Enzyme Digest Protocol