NYMU Taipei/Lab Notes/2007 9 16
From 2007.igem.org
(Difference between revisions)
(→Gel separation) |
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**10x EcoRI buffer: 2ul | **10x EcoRI buffer: 2ul | ||
**Total: 20ul | **Total: 20ul | ||
- | *Enzyme digestion: D-term with EcoRI | + | *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) | **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 | ||
Line 14: | Line 14: | ||
**H2O: 6ul | **H2O: 6ul | ||
**Total: 20ul | **Total: 20ul | ||
+ | |||
==Gel separation== | ==Gel separation== | ||
* 1% gel | * 1% gel |
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
- CinR+HSL (1~4): 14ul
- 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
- 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