|
Raphaël
Ethanol precipitation of the term/attC/mRFP (gel purification) to concentrate the sample.
Following this protocol : [1]
Elution with 20µL of water
Aleksandra
Miniprep
10 mL of culture, following Eric's advices : skip the step with PB buffer, thoroughly wipe the tube and the column after wash with PE buffer, elution with 50µL of water at 55°C.
- pSulib attC
- pSulib term/attC/mRFP
- TOP10 pSB1A2 pLux (R0062)
- TOP10 pSB1A2 RBS/GFP/term (E0240)
- TOP10 pSB1A2 pLux/GFP (1rst colony on the plate)
- TOP10 pSB1A2 pLux/GFP (2nd colony on the plate)
- TOP10 pSB1A2 pLux/GFP (3rd colony on the plate)
- TOP10 pSB1A2 pLux/GFP (4th colony on the plate)
Nanodrop quantification of DNA amounts in our samples
| Sample name |
260/280 |
260/230 |
ng/µl
|
|
|
|
|
|
|
|
|
| attC |
2.00 |
1.89 |
142.0
|
| term/attC/mRFP |
1.98 |
2.14 |
163.8
|
| pLux |
1.94 |
2.32 |
217.5
|
| GFP |
1.92 |
2.37 |
274.1
|
| pLux/GFP 1 |
1.93 |
2.30 |
208.7
|
| pLux/GFP 2 |
1.94 |
2.23 |
260.6
|
| pLux/GFP 3 |
1.95 |
2.26 |
264.3
|
| pLux/GFP 4 |
1.91 |
2.31 |
326.2
|
| term/attC/mRFP (ethanol prec.) |
1.27 |
0.12 |
5.2
|
Aleksandra and Raphaël
Restriction digest, incubation at 37°C for 2h.
- 20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x
- 20µl RBS/GFP/term + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x
- 20µl pLux + 1µl SpeI + 1µl PstI + 2.5µl buffer4 10x + 0.25µl BSA 100x
- 20µl attC + 1µl SpeI + 1µl PstI + 2.5µl buffer4 10x + 0.25µl BSA 100x
- 5µl pLux/GFP 1 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
- 5µl pLux/GFP 2 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
- 5µl pLux/GFP 3 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
- 5µl pLux/GFP 4 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
Aleksandra and Théotime
Gel electrophoresis (1.5% agarose, 50V)
- attC : 25µl + 5µl lb
- Ladder 1Kb
- term/attC/mRFP : 25µl + 5µl lb
- -
- pLux : 25µl + 5µl lb
- -
- GFP : 25µl + 5µl lb
- -
- pLux/GFP 1 : 20µl + 5µl lb
- pLux/GFP 2 : 20µl + 5µl lb
- pLux/GFP 3 : 20µl + 5µl lb
- pLux/GFP 4 : 20µl + 5µl lb
Gel purification with the Quiagen kit, elution in 50µl warm water.
Nanodrop quantification of DNA amounts in our samples
| Sample name |
260/280 |
260/230 |
ng/µl
|
|
|
|
|
|
|
|
|
| attC (gel purif.) |
2.67 |
0.03 |
5.0*
|
| term/attC/mRFP (gel purif.) |
2.53 |
0.05 |
3.9*
|
| pLux (gel purif.) |
2.18 |
0.15 |
11.0
|
| GFP (gel purif.) |
1.84 |
0.25 |
35.0
|
*These samples have no DNA : no peak at 260nm on the Nanodrop.
The yields of DNA are following : 8.8% for attC, 6% for term/attC/mRFP, 13% for pLux, 32% for RBS/GFP/term
Ligation
50ng of the vector and 150ng of insert in about 10μL of total volume. Ligation at 16°C overnight.
- 15μL GFP (E0240) + 1.5μL pLux (R0062) + 2μL buffer 10x + 0.5μL T4 ligase + 1µl water
Overnight culture of the transformations from 07/27 (colonies on all the plates)
10mL LB + Amp, 2 colonies for each transformation
- TetA(C) + stand.RBS
- RBS/GFP/term + pLux
- weakRBS/LuxR + Pmed
- weakRBS/LuxR + Pstr
- medRBS/LuxR + Pmed
- medRBS/LuxR + Pstr
- strRBS/LuxR + Pmed
- strRBS/LuxR + Pstr
- LacZ + weak RBS
- LacZ + stand RBS
- LuxI + term
Overnight culture from the glycerol stock
10mL LB
- 2x attC
- 2x term/attC/mRFP
Overnight culture of the transformation from 07/21, 5 colonies
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Memo-Cell
