NYMU Taipei/Lab Notes/2007 9 16

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Revision as of 15:01, 9 October 2007

Enzyme Digestion for CinR+HSL

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)

2nd digestion of D-term

D-term EcoRI + XbaI

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

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