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Léa and Théotime
Glycerols with 15%glycerol (375µl of 60% glycerol + 1125µl culture), all made in double for -20°C and -80°C:

• 16 cultures from the attC/term/attC/mRFP transformation plate from 31/07

Minipreps
Elution in 35µl of warm water

• attC
• term/attC/mRFP
• 16 cultures from the attC/term/attC/mRFP transformation plate from 31/07

Restriction digests
Preparative digests (20µl miniprep + 2x1µl enzymes + 2.5µl buffer + 0.25µl BSA)

• attC with SpeI+PstI (buffer2)
• term/attC/mRFP with XbaI+PstI (buffer3)

Verification digests (10µl miniprep + 6µl water + 2x1µl enzymes + 2µl buffer4 + 0.2µl BSA)

• attC/term/attC/mRFP (miniprep from colony #1 to #16) with EcoRI-HF+PstI

Gel electrophoresis (1.5%agarose w/v, 50V)
20µl restriction digest + 5µl 5x loading buffer

• ladder 1Kb
• attC/term/attC/mRFP #1 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #2 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #3 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #4 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #5 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #6 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #7 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #8 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #9 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #10 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #11 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #12 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #13 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #14 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #15 (digested with EcoRI-HF+PstI for verification)
• attC/term/attC/mRFP #16 (digested with EcoRI-HF+PstI for verification)

<a href="/wiki/Image:Gel0208_1.jpg" class="image" title="Image:gel0208_1.jpg"><img alt="Image:gel0208_1.jpg" src="/images/9/93/Gel0208_1.jpg" width="768" height="574" border="0" /></a>
Unfortunately, the ladder was added in every well instead of the loading buffer...

• term/attC/mRFP with XbaI+PstI
• -
• attC with SpeI+PstI
• -
• -
• ladder 1Kb

<a href="/wiki/Image:Gel0208_2.jpg" class="image" title="Image:gel0208_2.jpg"><img alt="Image:gel0208_2.jpg" src="/images/2/21/Gel0208_2.jpg" width="768" height="574" border="0" /></a>

Aleksandra

Restriction digests

Verification digests (5µl miniprep + 11µl water + 2x1µl enzymes + 2µl buffer2 + 0.2µl BSA)

• weak RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
• medium RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
• standard RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
• weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
• standard RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
• weak RBS/TetA(C) with EcoRI-HF+PstI
• medium RBS/TetA(C) with EcoRI-HF+PstI
• standard RBS/TetA(C) with EcoRI-HF+PstI
• weak RBS/LacZ alpha with EcoRI-HF+PstI
• standard RBS/LacZ alpha with EcoRI-HF+PstI
• LuxI/term with EcoRI-HF+PstI

Gel electrophoresis (1.5%agarose w/v, 50V)
20µl restriction digest + 5µl 5x loading buffer

• -
• weak RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
• weak RBS/LacZ alpha with EcoRI-HF+PstI
• medium RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
• medium RBS/TetA(C) with EcoRI-HF+PstI
• standard RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
• standard RBS/TetA(C) with EcoRI-HF+PstI
• ladder 1Kb
• ladder 100bp
• LuxI/term with EcoRI-HF+PstI
• -
• weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
• weak RBS/LacZ alpha with EcoRI-HF+PstI
• standard RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
• standard RBS/LacZ alpha with EcoRI-HF+PstI
• -
• attC (miniprep from 08/02)

<a href="/wiki/Image:Ge0208_3.jpg" class="image" title="Image:ge0208_3.jpg"><img alt="Image:ge0208_3.jpg" src="/images/a/a1/Ge0208_3.jpg" width="768" height="574" border="0" /></a>
This experiment indicates that the plasmid containing LuxI+term is not good (the length of the insert is 200bp instead of 700bp !)

Raphaël

Transformation of Turbo cells with yesterday's ligations using the Turbo cell transformation protocol:

• stand.RBS/TetA(C)/LuxI/term/pLux/GFP
• med.RBS/TetA(C)/LuxI/term/pLux/GFP
• weak.RBS/TetA(C)/LuxI/term/pLux/GFP
• stand.RBS/LacZα/LuxI/term/pLux/GFP
• weakRBS/LacZα/LuxI/term/pLux/GFP
• control 1 : pLux/GFP (vector only)
• control 2 : stand.RBS/TetA(C)/LuxI/term (insert only)

Making Amp plates
30 plates

DNA quantification in the minipreps. Measured with the Nanodrop

Sample name	260/280	260/230	ng/µl
attC/term/attC/mRFP #1	2.10	2.26	148.2
attC/term/attC/mRFP #2	2.01	2.13	95.6
attC/term/attC/mRFP #3	2.06	2.17	105.1
attC/term/attC/mRFP #4	2.06	2.17	91.1
attC/term/attC/mRFP #5	2.06	2.17	140.5
attC/term/attC/mRFP #6	2.09	2.06	109.9
attC/term/attC/mRFP #7	2.09	1.94	71.5
attC/term/attC/mRFP #8	2.10	2.18	85.9
attC/term/attC/mRFP #9	2.07	2.11	104.3
attC/term/attC/mRFP #10	1.72	0.76	36.3
attC/term/attC/mRFP #11	1.61	0.93	33.4
attC/term/attC/mRFP #12	2.08	2.06	135.5
attC/term/attC/mRFP #13	2.02	1.76	139.4
attC/term/attC/mRFP #14	2.09	2.03	96.8
attC/term/attC/mRFP #15	2.05	1.85	142.7
attC/term/attC/mRFP #16	2.07	2.00	147.4
attC	2.11	2.03	130.5
term/attC/mRFP	2.15	2.34	130.4