NYMU Taipei/Lab Notes/2007 9 16

From 2007.igem.org

(Difference between revisions)
(Enzyme Digestion)
(Enzyme Digestion)
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**10x EcoRI buffer: 2ul
**10x EcoRI buffer: 2ul
**Total: 20ul
**Total: 20ul
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*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)
+
*Enzyme digestion: D-term with EcoRI
-
**D-term: 11ul (損失因子10, 目標重量300ng)
+
** 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
*** 300(ng) * 10 = 3(ug) = 0.27 (ug/uL) * X(uL), X = 3/0.27 = 11.11111111
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**EcoRI: 1ul
+
** EcoRI: 1ul
-
**10x EcoRI buffer: 2ul
+
** 10x EcoRI buffer: 2ul
-
**H2O: 6ul
+
** H2O: 6ul
-
**Total: 20ul
+
** Total: 20ul
==Gel separation==
==Gel separation==

Revision as of 09:16, 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
  • 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