Team:Glendale CC AZ/Protocols/Ligation

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Ligation Protocol

Protocol from: http://parts.igem.org/Help:Protocols/Ligation

Materials 

  -digested plasmid backbone
  -equimolar amount of EcoRI-HF SpeI digested fragment
  -equimolar amount of XbaI PstI digested fragment
  -T4 DNA ligase buffer
  -T4 DNA ligase
  -dH2O

Procedure

1. Add 2µL of digested plasmid backbone

2. Add equimolar amount of digested Part 1

3. Add equimolar amount of digested Part 2

4. Add 1µL T4 DNA ligase buffer.

5. Add 0.5µL T4 DNA ligase.

6. Add water to 10µL.

7. Place in pre-programmed thermal cycler at 16°C for 30 minutes then at 80°C for 20 minutes.

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