Team:Carnegie Mellon/Protocols

Protocols.

How we did the things we did.

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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.
MiniPrep
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 Digestl
Restriction Enzyme Digest
Procedure:
  1. Prepare the following restriction enzyme digestion solution for J23108, J23109, J23111.
    2. Reagent Amount (µL)
    10X Fast Digest Buffer 2
    Plasmid DNA 12
    Restriction Enzyme XbaI 1
    Restriction Enzyme SpeI 1
    Water (to bring up to volume) 2
    Total Volume 18
  2. Digest at 37 °C for 1 hour.
  3. Follow the Life Technologies Restriction Enzyme Digest Protocol.
Agarose Gel Electrophoresis
Aragose Gel Electrophoresis
Procedure:
  1. 1% aragose gel made of:
      50 mL 0.5X TAE
      0.5g agarose
      2.5 µL ethidium bromide
  2. Run at 114V.
Gel Extraction
Gel Extraction
Procedure:
  1. Make 250-750 µl binding buffer (depending on mass of gel cut-out).
  2. Incubated at 42℃ until gel is melted.
  3. Follow Thermo Scientific's GeneJET Gel Extracton Protocol
Ligation
Ligation
Procedure:
  1. Make the following ligation reaction.
    2. Reagent Amount (µL)
    Promoter DNA 1
    Insert DNA 7
    Ligation Buffer 1
    Ligation Enzyme 1
    Total Volume 10
  2. Ligate for 10 minutes at room temperature.
  3. Put on ice until ready for transformation.
Transformation
Transformation
Procedure:
  1. Follow iGEM's Transformation Protocol