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

  PCR Block Settings:

  1. Initial Denaturation: 2.5 min @ 95°C 2. Denaturation: .5 min @ 95°C 3. Annealing: .5 min @ primer annealing temperature 4. Extension: 1 min @ 72°C Repeat steps 2-4 30 times 5. Final Extension: 3 min @ 72°C 6. Final Hold: indefinitely @ 4°C