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Protocols

Plasmids transformation :
Add 20 ng of plasmid to 100 µL of competent cells thawed in ice
Incubate 30-45 min in ice
Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
Incubate 5 min in ice
Add 900 µL of LB
Incubate 1 hour at 37°C with agitation
Spread 100 µL on LB limp (with antibiotic)
To make negative control, follow the same procedure but without add plasmids and spread 300 µL
Ligation transformation:
Add 20 ng of ligation’s product to 100 µL of competent cells thawed in ice
Incubate 30-45 min in ice
Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
Incubate 5 min in ice
Add 900 µL of LB
Incubate 1 hour at 37°C with agitation
Centrifuge 5 min at 5000 rpm
Eliminate 850 µL of medium
Suspend the pellet
Spread 150 µL on LB limp (with antibiotic)
To make negative control, follow the same procedure but without add plasmids and spread 300 µL

Preparation of competent bacteria cells
Make a culture of your bacteria in LB medium and let it grow until bacteria are in exponential phase (OD600 = 0.5).
Cells are cold centrifuge 10 min at 3500 rpm.
The pellet is slowly suspended in 80 mL of Tfb1 buffer (300mM KOAc, 0.05M MnCl2, 0.1M KCl, 0.01M
CaCl2, 15% Glycerol (see next section “Preparation of Tbf1 and Tbf2 buffer”).
After another 5 min cold centrifugation at 3500 rpm, the pullet is suspended in 8 mL of Tbf2 Buffer
(0.01mM NaMOPS pH 7, 0.075M CaCl2, 0.01M KCl, 15% Glycerol)
Incubate 15 min in ice. Aliquot 200µL of cell suspension in sterile Eppendorf tubes. The cell
suspension is conserved at -80°C.

Preparation of Tbf1 and Tbf2 buffer
For 200 mL of culture:
Preparation of 80 mL of Tbf1 Buffer:

KAc 1M	2.4 mL
MnCl2 0.5M	8 mL
KCl 1 M	8 mL
CaCl2 0.1M	8 mL
Gly 80%	15 mL
H2O	38.6 mL

Preparation of 8 mL of Tbf2 Buffer:

NaMOPS 0.2M	400 µL
CaCl2 0.1M	6 mL
KCl 1 M	8 mL
Gly 80%	1.5 mL
KCl 1M	80 µL
H2O	500 µL

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50% glycerol	6.25 mL Glycerol 80%
20 mM EDTA	0.4 mL EDTA 0.5 M
0.05% Bromophenol blue	≈0.05g Bromophenol blue
0.05% Xylene cyanol	≈0.05g xylene cyanol
1% SDS	1 mL SDS 10%

Recipe for SES 4X:

50% glycerol	6.25 mL Glycerol 80%
20 mM EDTA	0.4 mL EDTA 0.5 M
0.05% Bromophenol blue	≈0.05g Bromophenol blue
0.05% Xylene cyanol	≈0.05g xylene cyanol
1% SDS	1 mL SDS 10%

Digestion (verification) protocol DNA Between 50 and 100 ng EcoRI-HF 0.2 µL PstI 0.2 µL 10X NEBuffer 2 2 µL 100X BSA 0.2 µL H2O QS 20 µL Incubate all digest reactions at 37°C for 1 hour and then add 3 µL of SES 4X and migrate 30 min at 150V on a 1% agarose gel. Digestion protocol BioBrick Assembly Kit Upstream part : Upstream part plasmid 500 ng EcoRI-HF 1 µL SpeI 1 µL 10X NEBuffer 2 5 µL 100X BSA 0.5 µL H2O To 50 µL Downstream part : Downstream part plasmid 500 ng XbaI 1 µL PstI 1 µL 10X NEBuffer 2 5 µL 100X BSA 0.5 µL H2O To 50 µL Destination plasmid Destination plasmid 500 ng EcoRI-HF 1 µL PstI 1 µL DpnI 1 µL 10X NEBuffer 2 5 µL 100X BSA 0.5 µL H2O To 50 µL Incubate the three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes. Ligation protocol BioBrick Assembly Upstream part digestion 2 µL Downstream part digestion 2 µL Destionation plasmid digestion 2 µL 10X T4 DNA ligase buffer 2 µL T4 DNA ligase 1 µL H2O 11 µL Incubate at RT for 1 hour.

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