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Stéphane and Aleksandra
Verification of the transformations from 07/23 and 07/22 and if successful preparing them for future ligation
Restriction digest of the minipreps from 07/25 and 07/23, incubation at 37°C for 2h
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
- medium RBS (B0032) - TetA(C) (J31007), transformation on 07/23
- pLux (R0062) - RBS/GFP/term (E0240), transformation on 07/22
40µl miniprep + 4.7µl Buffer3 10x + 0.5 µl BSA 100x + 2µl XbaI + 2µl PstI (total of 50µl) = double
- weak RBS (B0031) - LuxR (C0062), transformation on 07/23
- medium RBS (B0032) - LuxR (C0062), transformation on 07/23
- strong RBS (B0034) - LuxR (C0062), transformation on 07/23
Gel electrophoresis (agarose 1.5% w/v, 50V)
- weak RBS-TetA(C)
- ladder
- medium RBS-TetA(C)
- -
- weak RBS-LuxR
- weak RBS-LuxR
- -
- medium RBS-LuxR
- medium RBS-LuxR
- -
- strong RBS-LuxR
- strong RBS-LuxR
 
The transformations from 07/23 have worked, we have the inserts of the expected size : 1.2kb for RBS+TetA(C) and 0.8kb for RBS+LuxR. We can purify these inserts to use them in further ligations.
However, the transformation of pLux-RBS/GFP/term from 07/22 didn't work (see the gel made by Théotime further on this page)
Gel extraction
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
|
| | | |
|
| | | |
|
| weak RBS-TetA(C) | 1.62 | 0.11 | 11.8
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| medium RBS-TetA(C) | 1.90 | 0.09 | 22.8
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| | | |
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| weak RBS-LuxR | 1.76 | 0.38 | 32.3
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| medium RBS-LuxR | 1.89 | 0.08 | 36.8
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| standard RBS-LuxR | 1.80 | 0.16 | 40.7
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Théotime and Raphaël
Restriction digest of the biobricks we might need again if the 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
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
|
| | | |
|
| | | |
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| LacZ alpha | 1.94 | 0.02 | 9.0
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| LuxI | 1.82 | 0.05 | 10.2
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| | | |
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| | | |
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| LuxR | 1.87 | 0.13 | 36.6
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| TetA(C) | 1.88 | 0.37 | 71.8
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| RBS/GFP/term | 1.72 | 0.15 | 15.1
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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. All of the vector DNA (RBSs, Pm, Ps) are diluted 1/5. Incubation at 16°C overnight.
- 2µl TetA(C) + 0.9µl stand.RBS + 1µl buffer + 0.5µl ligase + 5.5µl water
- 10µl RBS/GFP/term + 3.6µl pLux + 1µl buffer + 0.5µl ligase + 4µl water
- 5µl weakRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase
- 5µl weakRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 0.8µl water
- 4µl medRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase + 1µl water
- 4µl medkRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 1.8µl water
- 4µl strRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase + 1µl water
- 4µl strRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 1.8µl water
- 13µl LacZ + 0.7µl weak RBS + 2µl buffer + 0.5µl ligase + 3.8µl water
- 13µl LacZ + 0.9µl stand RBS + 2µl buffer + 0.5µl ligase + 3.6µl water
- 15µl LuxI + 2.9µl weak RBS + 2µl buffer + 0.5µl ligase
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Memo-Cell
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