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
- The genes for Taq Ligase and T5 exonuclease were designed synthesized in a factory plasmid by GeneArt
- Prepare 100 mL stocks of BL21(DE3) competent cells (stored at -80°C)
- Allow to unthaw, and add 2 uL DNA (using 2uL water as a control) to competent cell cultures.
- Let sit on ice for ten minutes
- Heat-shock cells for 45 seconds in 42°C water bath
- Return cells to ice for two minutes
- Allow cells to recover by adding 900 uL LB and putting into 37°C shaking incubator for 1 hour
- Plate 100 uL on Kan(50) LB agarose plates and incubate for 16 hours at 37°C
- 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:
- Pick a colony from the cell culture using a sterile toothpick
- Streak out on Kan(50) lysogeny broth (LB) agarose
- Place in the 25uL Reaction Mix and spin toothpick for a few seconds
- 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
PCR Block Settings: