Team:Northwestern/ligation

Phosphorylation and Ligation

• Phosphorylate, ligate and transform immediately after each other
  
• Modified Phosphorylation Procedure:
• 150 ng DNA
• 3 uL T4 ligase buffer
• 1 uL T4 PNK
• Water to 30 uL
• Run at 37°C for 1 hour
  
• Ligation: (into storage vectors-pSB1K3)
• 20 uL reaction volume:
• 50 ng backbone (linearized pSB1K3 at 25 ng/uL from iGEM)
• *Use NEBioCalculator, 3:1 insert:backbone molar ratio
• 2 uL T4 ligase buffer
• 1 uL T4 ligase
• Water to 20 uL
• Run for 15 minutes (max 30) at room temperature
• Heat to inactivate at 80°C for 20 minutes or transform immediately