NYMU Taipei/Lab Notes/2007 9 16
From 2007.igem.org
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** CinR+HSL insert size = 1.53 Kb | ** CinR+HSL insert size = 1.53 Kb | ||
** D-term vector size = 3.284 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 |
Revision as of 09:13, 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
- CinR+HSL (1~4): 14ul
- Enzyme digestion: D-term with EcoRI
- 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
- D-term: 11ul (損失因子10, 目標重量300ng)
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