Team:Aix-Marseille/Protocols
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.
The sequences are already prepared like BioBrick Assembly, they also have the prefixe and suffixe
This protocol is made to prepare the DNA, digest, ligate and transform it.
- 1- Resuspend gBlocks Gene fragment to a final concentration of 10 ng/µL in TE or AE (elution buffer). The tube contain 1000 ng of gBlocks Gene fragment so you have to add 100µL of AE
- 2- Mix well, a vortex shall be used
Digestion E/P
Digest 100 ng in 50µL| Component | 50 µL of reaction |
| DNA | 10 µL |
| Net buffer 2.1 | 5 µL |
| H2O | 33 µL |
| EcoRI | 1 µL |
| Pst I | 1 µL |
1- Incubate for 45 min at 37°C 2- Incubate 20 min at 80°C (inactivation of restriction enzyme)
| Component | 50 µL of reaction |
| DNA | 10 µL |
| Net buffer 2 (10X) | 5 µL |
| BSA (100X) | 0.5 µL |
| H2O | 33 µL |
| EcoRI | 1 µL |
| Pst I | 1 µL |
Ligation
- 1- Ligate 50 ng vector with a 3X molar excess of gBlocks Gene fragments in fresh T4 DNA ligase buffer diluted to 1X and 400 U T4 DNA ligase for a total volume of 20 µL
| Component | 20 µL of reaction |
| PSBIC3 (or other vector) at 12.5 ng/µL | 2 µL |
| insert digested (at 37.5 ng/µL) | 12 µL |
| T4 ligase buffer | 2 µL |
| T4 ligase 400U | 1 µL |
| H2O | 3 µL |
- 1- Incubate 2h at room temperature
Transformation
- 1- Add 10 µL of ligation’s product to 100 µL of competent cells thawed in ice (TGI cells)
- 2- Incubate 45 min in ice
- 3- Thermal shock: put tubes in the thermomixer at 42°C during 2 min
- 4- Incubate 5 min in ice
- 5- Add 900 µL of LB
- 6- Incubate 1 hour at 37°C with agitation
- 7- Centrifuge 5 min at 5000 rpm
- 8- Eliminate 850 µL of supernatant
- 9- Suspend the pellet in the 150µL of remaining medium
- 10- Spread 150 µL on LB limp (with antibiotic)
To make negative control, follow the same procedure but without add plasmids and spread 300 µL
Take the EconoTaq PLUS 2X Master Mix only when all your tubes are ready and keep it on ice all the time you need it.
Add the EconoTaq PLUS 2X Master Mix at the end, when your tubes contain all the other components..
| 25 µL Reaction | 50 µL Reaction | 100 µL Reaction | Final concentration | |
| EconoTaq PLUS 2X Master Mix | 12.5 µL | 25 µL | 50 µL | 1X |
| Forward Primer (100 pmol/µL) | 0.5 µl | 0.5 µL | 1 µL | 1 pmol/µL (1µM) |
| Reverse Primer (100 pmol/µL) | 0.5 µL | 0.5 µL | 1 µL | 1 pmol/µL (1µM) |
| DNA Template* | 0.5 µL | 1 µL | 1 µL | |
| Water nuclease-free | 11 µL | 23 µL | 47 µL |
*One colony in 50 µL of Water.15-20 min at 80°C. 5 min at 96°C.
| Cycling Step | Temperature | Time | Number of cycles |
| Initial denaturation | 98°C | 3 min | 1 |
| Denaturation | 95°C | 10 sec | X 27 |
| Annealing | 50-65°C | 40 sec | X 27 |
| Extension | 72°C | 1 min/kb | X 27 |
| Hold | 16°C | Indefinitely |
Take the Q5 High Fidelity DNA Polymerase only when all your tubes are ready and keep it on ice all the time you need it.
Add the Q5 High Fidelity DNA Polymerase at the end, when your tubes contain all the other.
| Component | 50 µL reaction |
| 5X Q5 Buffer | 10 µL |
| dNTP (10 µM) | 1 µL |
| Primer Forward 10 µM | 2.5 µL |
| Primer Reverse 10 µM | 2.5 µL |
| DNA | 1 colony* or 1 µL |
| Q5 Polymerase | 0.5 µL |
| Water | To 50 µL |
*One colony in 50 µL of Water. 15-20 min at 80°C. 5 min at 96°C.
| Cycling Step | Temperature | Time | Number of cycles |
| Initial denaturation | 98°C | 10 min if DNA is from a colony else 3 min |
1 |
| Denaturation | 98°C | 10 sec | X 27 |
| Annealing | 55°C | 20 sec | X 27 |
| Extension | 72°C | 30 sec/kb | X 27 |
| Hold | 16°C | Indefinitely |
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 !
