Team:Aix-Marseille/Protocols2
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
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.
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 |
| 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 |
| 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% |
| 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 | To 20 µL |
150V on a 1% agarose gel.
Upstream part :
| Upstream part plasmid | 500 ng |
| EcoRI-HF | 1 µL |
| PstI | 1 µL |
| 10X NEBuffer 2 | 2.5 µL |
| 100X BSA | 0.5 µL |
| H2O | To 50 µL |
Downstream part :
| Downstream part plasmid | 500 ng |
| Xba I | 1 µL |
| Spe I | 1 µL |
| 10X NEBuffer 2 | 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 | 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.
| Upstream part digestion | 2 µL |
| Dowstream part digestion | 2 µL |
| Destination plasmid | 2 µL |
| 10X T4 DNA ligase buffer | 2 µL |
| T4 DNA ligase | 1 µL |
| H2O | 11 µL |
Incubate at RT for 1 hour.
![](http://i.imgur.com/5kMiAZ3.png")
![](http://i.imgur.com/a51RQFP.png")
For each construction the biobricks corresponding is RBS (B0034), GFP (E0040) and Terminator (B0010-B0012)
All the construction was introduced in the plasmid PSB1C3 and cloning in E.Coli DH5α
All the construction was introduced in the plasmid PSB1C3 and cloning in E.Coli DH5α
Day 1: Streak on plates clones containing constructions.
Day 2: Starters were prepared in 3 mL LB media. One colony of each construction was inoculated. We did biological triplicates.Starters were incubated during 16-18 hours at 300 rpm and 37°C with shaking.
Day 3: Measurement
1)Calibration: Dilution of Atto488 in triplicates with PBS1X and LB
2) Preparation samples
Each construction was biologically triplicate.
OD of each sample was adjusted at 0.5 diluted into LB media (0.475< OD <0.525).
200µl of each sample were put into a 96-wells plate. A triplicate of the measure was also done for each biological triplicate.
3) Parameters
Excitation Wavelength 488 nm
Emission Wavelength 520 nm
Excitation Bandwidth 9 nm
Emission Bandwidth 20 nm
Gain 100 Manual
Number of Flashes 25
Integration Time 20 µs
2) Preparation samples
Each construction was biologically triplicate.
OD of each sample was adjusted at 0.5 diluted into LB media (0.475< OD <0.525).
200µl of each sample were put into a 96-wells plate. A triplicate of the measure was also done for each biological triplicate.
3) Parameters
| Label: GFP | ||
| Mode | Fluorescence Top Reading | |
| Clarification of the lysis | Shake gently until the blue disappear | Centrifuge 5-10 min at 11.000 g |
| Bind DNA | Load supernatant on a new column with a discard tube below | Centrifuge 1 min at 11.000 g |
| Wash silica membrane | Discard the content of the discard tube and replace it below the column | (optional: Add 500 µL of Buffer AW and incubate 5 min at RT) Add 600 µL of Buffer A4 Centrifuge 1 min at 11.000 g |
| Dry silica membrane | discard the content of the discard tube and replace it below the column | Centrifuge 2 min at 11.000 g |
| Elute DNA | Place a 1.5 mL Eppendorf tube below the column | Add 50 µL of Buffer AE Incubate 1 min at RT Centrifuge 1 min at 11.000g |
This is the plasmid purification kit of the Igem-AMU team of 2014.
Use the kit with gloves and without fire.
| Cultivate and harvest bacterial cells | Harvest the bacterial cells in a 2 mL tube | Centrifuge 30 sec at 11.000 g |
| Cell lysis | Add 250µL of Buffer A1 and suspend the cells Then Add 250µL of Buffer A2 Incubate 5 min at RT Add 300 µL of Buffer AE3 | |
| Clarification of the lysis | Shake gently until the blue disappear | Centrifuge 5-10 min at 11.000 g |
| Bind DNA | Load supernatant on a new column with a discard tube below | Centrifuge 1 min at 11.000 g |
| Wash silica membrane | Discard the content of the discard tube and replace it below the column | (optional: Add 500 µL of Buffer AW and incubate 5 min at RT) Add 600 µL of Buffer A4 Centrifuge 1 min at 11.000 g |
| Dry silica membrane | discard the content of the discard tube and replace it below the column | Centrifuge 2 min at 11.000 g |
| Elute DNA | Place a 1.5 mL Eppendorf tube below the column | Add 50 µL of Buffer AE Incubate 1 min at RT Centrifuge 1 min at 11.000g |
Nanodrop measurement:
Add 2µL of buffer AE, close the machine and open it again, then clean up and down with paper.
Add 2µL of buffer AE, close and mesure for the blank, then clean up and down with paper.
Add 2µL of your sample and close it. Don’t forget to note your concentration !
Clean your sample with paper, then clean up and down with AE
Don’t put your finger on the paper when you are cleaning, you could contaminate the sample with YOUR DNA !
