IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/26
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Stéphane and Aleksandra
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Théotime
Restriction digest of the biobricks we might need again ifthe transformations don't work
20µl miniprep + 2.5µl Buffer 10x + 0.25 µl BSA 100x + 1µl enzyme1 + 1µl enzyme2 (total of 25µl)
- RBS/GFP/term (E0240) (XbaI + PstI, B3)
- RBS/LuxI (K081008) (EcoRI-HF + SpeI, B4)
40µl miniprep + 4.7µl Buffer 10x + 0.5 µl BSA 100x + 2µl enzyme1 + 2µl enzyme2 (total of 50µl) = double
- LuxR (C0062) (XbaI + PstI, B3)
- LacZ alpha (XbaI + PstI, B3)
- TetA(C) (XbaI + PstI, B3)
Gel electrophoresis (1.5% agarose w/v, 50V)
20µl digestion product + 5µl lb 6x
- ladder 1kb
- RBS/GFP/term
- -
- TetA(C)
- TetA(C)
- -
- LuxR
- LuxR
- -
- -
- -
- pLux-GFP (ligation product purified by Aleksandra and Stéphane)
Gel electrophoresis (3.0% agarose w/v, 50V)
20µl digestion product + 5µl lb 6x
- ladder 0.1kb
- -
- LacZ alpha
- LacZ alpha
- -
- LuxI
All of the restictions worked (except for the verification of the ligation pLux-GFP).
Gel extraction>/u>
Elution from the column with 30µl of warm water (50°C). Samples of the same part are collected into 1 tube.
Nanodrop quantification of DNA amounts in the gel extraction samples
Sample name | 260/280 | 260/230 | ng/µl |
LacZ alpha | 1.94 | 0.02 | 9.0 |
LuxI | 1.82 | 0.05 | 10.2 |
LuxR | 1.87 | 0.13 | 36.6 |
TetA(C) | 1.88 | 0.37 | 71.8 |
RBS/GFP/term | 1.72 | 0.15 | 15.1 |
Ligation using around 150ng of the insert and 50µg of the vector, in 10µl of total volume if possible, or in a minimum volume if >10µl. Incubation at 16°C overnight.
- 2µl TetA(C) + 0.9