From 2007.igem.org

Enzyme Digestion

• Enzyme digestion: CinR+HSL (with RBS) with EcoRI, SpeI
  • CinR+HSL (1~4): 14ul
    • 因為濃度未知, 所以用最大體積
  • EcoRI: 1ul (20,000 units/ml)
  • SpeI: 1ul (10,000 units/ml)
  • 10x EcoRI buffer: 2ul
  • Total: 20ul
  • 37° 2h
• Enzyme digestion: D-term with EcoRI
  • Original we need double digestion EcoRI and XbaI.
  • However, NEB recommend to perform the digestion in sequential manner.
  • Thus, we digest the EcoR1 first
  • D-term: 11ul (損失因子10, 目標重量300ng)
    • 300(ng) * 10 = 3(ug) = 0.27 (ug/uL) * X(uL), X = 3/0.27 = 11.11111111
  • EcoRI: 1ul
  • 10x EcoRI buffer: 2ul
  • H2O: 6ul
  • Total: 20ul
  • 37°C 2h
• Enzyme digestion: D-term E with XbaI
  • D-term E: 30ul
  • XbaI: 2ul
  • 10x 2 buffer: 5ul
  • 10x BSA: 5ul
  • H2O: 8ul
  • Total: 50ul
  • 37°C 2h

Gel separation

• 1% gel, TAE 1X, 30 min, 100v
  • 11 lanes (left most is marker)
  • each sample (24 uL) are separated into 2 lanes (12 uL for each lane)
    • CinR+HSL #1-#4 (lane 2-9) and D-term (lane 10-11)
• use 1Kb ladder
  • CinR+HSL insert size = 1.53 Kb
  • D-term vector size = 3.284 Kb
• 6X dye
  • total volume after digestion is 20uL
  • thus, the dye is 4uL
    • X/(20+X) = 1/6, X = 4
  • sample after dye addition is 20 + 4 = 24 uL

• Lane 1: 1kb ladder (5ul)
• Lane 2, 3: CinR+HSL 1 EcoRI, SpeI
• Lane 4, 5: CinR+HSL 2 EcoRI, SpeI
• Lane 6, 7: CinR+HSL 3 EcoRI, SpeI
• Lane 8, 9: CinR+HSL 4 EcoRI, SpeI
• Lane 10, 11: D-term EcoRI
• Comments
  • The concentration of CinR+HSL 1 is too low.
  • The insert of CinR+HSL 2 is wrong.
  • EcoRI digestion of D-term is incomplete, hence only the upper band is isolated for DNA extraction. (The lower band is probably the negative supercoiled form of the plasmid)

• Lane 1: 1kb ladder
• Lane 2, 3: D-term EcoRI, XbaI
• Lane 4, 5: Empty