IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/26: Difference between revisions
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<br><u>Verification of the transformations from 07/23 and 07/22 and if successful preparing them for future ligation</u> | <br><u>Verification of the transformations from 07/23 and 07/22 and if successful preparing them for future ligation</u> | ||
<br><u>Restriction digest of the minipreps from 07/25 and 07/23</u>, incubation at 37°C for 2h | <br><u>Restriction digest of the minipreps from 07/25 and 07/23</u>, incubation at 37°C for 2h | ||
20µl miniprep + 2.5µl Buffer3 10x + 0.25 µl BSA 100x + 1µl XbaI + 1µl PstI (total of 25µl) | <br>20µl miniprep + 2.5µl Buffer3 10x + 0.25 µl BSA 100x + 1µl XbaI + 1µl PstI (total of 25µl) | ||
*weak RBS (B0031) - TetA(C) (J31007), transformation on 07/23 | *weak RBS (B0031) - TetA(C) (J31007), transformation on 07/23 | ||
*medium RBS (B0032) - TetA(C) (J31007), transformation on 07/23 | *medium RBS (B0032) - TetA(C) (J31007), transformation on 07/23 | ||
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*strong RBS (B0034) - LuxR (C0062), transformation on 07/23 | *strong RBS (B0034) - LuxR (C0062), transformation on 07/23 | ||
<br><u>Gel electrophoresis</u> | <br><u>Gel electrophoresis (agarose 1.5% w/v, 50V)</u> | ||
*weak RBS-TetA(C) | *weak RBS-TetA(C) | ||
*ladder | *ladder | ||
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<font color=red>Théotime</font> | |||
<br><u>Restriction digest of the biobricks we might need again ifthe transformations don't work</u> | |||
<br>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) | |||
<br>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) | |||
<br><u>Gel electrophoresis (1.5% agarose w/v, 50V)</u> | |||
<br>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) | |||
[[Image:gel2607_2.jpg]] | |||
<br><u>Gel electrophoresis (3.0% agarose w/v, 50V)</u> | |||
<br>20µl digestion product + 5µl lb 6x | |||
*ladder 0.1kb | |||
*- | |||
*LacZ alpha | |||
*LacZ alpha | |||
*- | |||
*LuxI | |||
[[Image:gel2607_3.jpg]] | |||
__NOTOC__ | __NOTOC__ |
Revision as of 09:17, 27 July 2010
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Stéphane and Aleksandra
|
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