Team:Wisconsin-Madison/protocol

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Protocol

Expression and Purification of Enzymes

Solutions

Resuspension Buffer

  • 400 mM NaCl
  • 50 mM NaHPO4
  • 2.5% (v/v) glycerol
  • 15 mM Imidazole
  • 0.05% Triton X-100
  • pH 8.0

Taq Ligase Storage Buffer

  • 10 mM Tris-HCl
  • 50 mM KCl
  • 1 mM DTT
  • 0.1 mM EDTA
  • 200 μg/ml BSA
  • 50% v/v Glycerol
  • pH 7.4 @ 25°C

T5 Exonuclease Storage Buffer

  • 50 mM Tris-HCl
  • 100 mM NaCl
  • 1 mM DTT
  • 0.1 mM EDTA
  • 50% v/v Glycerol
  • 0.1% Triton® X-100
  • pH 7.5 @ 25°C

Transforming the Synthesized Plasmid

  1. The genes for Taq Ligase and T5 exonuclease were designed synthesized in a factory plasmid by GeneArt
  2. Prepare 100 mL stocks of BL21(DE3) competent cells (stored at -80°C)
  3. Allow to unthaw, and add 2 uL DNA (using 2uL water as a control) to competent cell cultures.
  4. Let sit on ice for ten minutes
  5. Heat-shock cells for 45 seconds in 42°C water bath
  6. Return cells to ice for two minutes
  7. Allow cells to recover by adding 900 uL LB and putting into 37°C shaking incubator for 1 hour
  8. Plate 100 uL on Kan(50) LB agarose plates and incubate for 16 hours at 37°C
  9. Determine which colonies have successfully taken up plasmid by cPCR

Colony PCR Protocol

Each tube:
  • 25uL PCR tube contents:
  • 1uL of Forwards and backwards Primers at 10uM concentration
  • 10.5uL Nuclease Free H20
  • 12.5uL goTaq
  • Cell Culture

Method:

  1. Pick a colony from the cell culture using a sterile toothpick
  2. Streak out on Kan(50) lysogeny broth (LB) agarose
  3. Place in the 25uL Reaction Mix and spin toothpick for a few seconds

  4. PCR Block Settings:
  5. 1. Initial Denaturation: 2.5 min @ 95°C
  6. 2. Denaturation: .5 min @ 95°C
  7. 3. Annealing: .5 min @ primer annealing temperature
  8. 4. Extension: 1 min @ 72°C
  9. Repeat steps 2-4 30 times
  10. 5. Final Extension: 3 min @ 72°C
  11. 6. Final Hold: indefinitely @ 4°C