NYMU Taipei/Lab Notes/2007 9 16

From 2007.igem.org

(Difference between revisions)
(Gel separation)
(Gel separation)
Line 29: Line 29:
** thus, the dye is 4uL
** thus, the dye is 4uL
*** X/(20+X) = 1/6, X = 4
*** X/(20+X) = 1/6, X = 4
 +
** sample after dye addition is 20 + 4 = 24 uL

Revision as of 09:19, 16 September 2007

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
  • 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

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)
  • 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