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Théotime
Restriction digest of the same minipreps as yestarday (concentration measured by nanodrop 07/22) using the same protocol as yesterday : 3µg of DNA per reaction.
- RBS/LuxI (K081008) : 32µl DNA + 4µl Buffer 4 10x + 0.4µl BSA 100x + 2µl EcoRI-HF + 2µl SpeI
- TetA(C) (J31007) in a triple volume : 60µl DNA + 7.5µl Buffer 3 10x + 0.8µl BSA 100x + 4µl XbaI + 4µl PstI
- LacZ alpha (I732006) in a triple quantity : : 41µl DNA + 5.5µl Buffer 3 10x + 0.6µl BSA 100x + 4µl XbaI + 4µl PstI
- LuxR (C0062), in triple : 59µl DNA + 7.5µl Buffer 3 10x + 0.7µl BSA 100x + 4µl XbaI + 4µl PstI
- attC : 111µl DNA + 13µl Buffer 2 10x + 1.3µl BSA 100x + 2µl SpeI +2µl PstI
- term/attC/mRFP : 91µl DNA + 11µl Buffer 3 10x + 1.1µl BSA 100x + 2µl XbaI + 2µl PstI
- pLux (R0062) : 49µl DNA + 6µl Buffer 2 10x + 0.6µl BSA 100x + 2µl SpeI + 2µl PstI
- RBS/GFP/term (E0240) : 26µl DNA + 3.5µl Buffer 3 10x + 0.4µl BSA 100x +2 µl XbaI + 2µl PastI
Stéphane
Transformation of TOP10 chemically competent E.coli with the overnight ligations from 07/22. Recuperation for 30 mins after the heat shock in SOC, then plating with 150µl of the transformmation on the ampicilin plate, incubation overnight at 37°C.
- TOP10 pSB1A2 weak RBS -TetA(C)
- TOP10 pSB1A2 med.RBS -TetA(C)
- TOP10 pSB1A2 weak RBS -LuxR
- TOP10 pSB1A2 med.RBS -LuxR
- TOP10 pSB1A2 stand.RBS -LuxR
Théotime and Aleksandra
Gel electrophoresis of the restriction digest products, only for the future inserts.
Agarose gel 0.8%, 50V to separate the big inserts
- TetA(C) (J31007) : 37µl + 7.4µl lb
- TetA(C) (J31007) : 37µl + 7.4µl lb
- ladder
- LuxR (C0062) : 37µl + 7.4µl lb
- LuxR (C0062) : 37µl + 7.4µl lb
- -
- term/attC/mRFP : 37µl + 7.4µl lb
- term/attC/mRFP : 37µl + 7.4µl lb
- term/attC/mRFP : 37µl + 7.4µl lb
- -
- RBS/GFP/term : 37µl + 7.4µl lb
- ladder
Agarose gel 3%, 50V to separate the small inserts
- ladder (Quick load 100bp ladder), 5µl
- LacZ alpha (I732006) : 27µl + 5.5µl lb
- LacZ alpha (I732006) : 27µl + 5.5µl lb
- -
- RBS/LuxI (K081008) : 40µl + 8µl lb
- RBS/LuxI (K081008) : 40µl + 8µl lb
Raphaël
Gel purification with the Quiagen kit.
All bands are eluted with 30μL of water.
Stéphane
DNA purification from the 07/23 minipreps using the PCR purification kit.
Elution with 30µl water. In the result we have 2x28µl of each biobrick that will serve us as a vector for the ligation:
Raphaël
Minipreps : 4 minipreps of a 5ml culture per biobrick, eluted with 30μL buffer and collected into 1 tube for a total volume of around 120μL:
- attC
- term/attC/mRFP
- attC-term/attC/mRFP (colony from the transformation plate of 07/21)
- pLux-RBS/GFP/term (colony from the transformation plate of 07/21)
Nanodrop to determine the amount of DNA in the miniprep samples and after gel purification and PCR purification:
| Sample name |
260/280 |
260/230 |
ng/μL
|
| attC(miniprep) |
1.85 |
2.09 |
66.6
|
| t-attC-mRFP (miniprep) |
1.85 |
2.17 |
78.3
|
| pLux-GFP (miniprep) |
1.86 |
2.38 |
174.8
|
| attC-t-attC-mRFP (miniprep) |
1.83 |
2.28 |
75.9
|
| attC (PCR purif.) |
1.70 |
1.32 |
20.0
|
| pLux (PCR purif.) |
1.79 |
1.88 |
70.3
|
| LuxI (gel purif.) |
1.71 |
0.05 |
5.1
|
| LacZ (gel purif.) |
2.26 |
0.02 |
3.1
|
| TetA(C) (gel purif.) |
1.92 |
0.08 |
10.8
|
| GFP (gel purif.) |
1.80 |
0.08 |
6.2
|
| t-attC-mRFP (gel purif.) |
1.40 |
0.01 |
1.6
|
| LuxR (gel purif.) |
1.66 |
0.06 |
3.4
|
Obviously, there is not enough DNA after gel purification to do a ligation...
Overnight culture
2x20mL per biobrick, the goal is to do minipreps on 10mL of pellet tomorrow (increase the initial amount of DNA >> increase the amount of DNA after gel purification !)
- LacZ alpha (I732006) - Amp
- RBS/GFP/term (E0240) - Amp
- LuxR (C0062) - Amp
- RBS/LuxI (K081008) - Amp
- TetA(C) (J31007) - Amp
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