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Aleksandra and Raphaël
Transformation of TOP10 cells (25µl/transformation) with the 07/26 ligations:
- TetA(C) + stand.RBS
- RBS/GFP/term + pLux
- weakRBS/LuxR + Pmed
- weakRBS/LuxR + Pstr
- medRBS/LuxR + Pmed
- medkRBS/LuxR + Pstr
- strRBS/LuxR + Pmed
- strRBS/LuxR + Pstr
- LacZ + weak RBS
- LacZ + weak RBS
- LuxI + weak RBS
Incubation at 37°C for 21h.
Aleksandra
Miniprep
4x10ml cultures of term/attC/mRFP, elution with 30µl EB from each column = 120µl DNA in total
Nanodrop quantification of DNA in the samples
| Sample name |
260/280 |
260/230 |
ng/µl
|
|
|
|
|
|
|
|
|
| term/attC/mRFP |
1.99 |
2.04 |
97.8
|
| term/attC/mRFP, concentrated |
1.82 |
2.01 |
114.8
|
We concentrated 60µl of the sample using the PCR purification kit, eluting in 30µl warm water, but the concentration wasn't successful enough. Anyway, we used the concentrated sample for the restriction digest.
Restriction digest, incubation at 37°C for 2h.
- 20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x
- 40µl attC (miniprep of 07/25) + 1µl SpeI + 1µl PstI + 4.7µl buffer3 10x + 0.45µl BSA 100x
Théotime
DNA purification from the restriction digest reaction using the PCR purification kit
- attC eluted with 30µl of water
Making amp plates
21 plate
Raphaël
Gel electrophoresis (1.5% agarose, 50V)
- ladder 1kb
- -
- term/attC/mRFP
- -
- -
- -
Gel purification and nanodrop to quantify DNA
| Sample name |
260/280 |
260/230 |
ng/µl
|
|
|
|
|
|
|
|
|
| attC (PCR purif.kit) |
1.67 |
1.01 |
6.2
|
| term/attC/mRFP (gel purif.) |
2.01 |
0.05 |
14.4
|
We often have very low 260/230 values : probably contamination with the solvents.
Ethanol precipitation of the term/attC/mRFP (gel purification) to concantrate the sample.
60µl ethanol + 2.4µl sodium acetate 3M + 24µl term/attC/mRFP, incubation at -20°C overnight.
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Memo cell
