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Team:Aix-Marseille/Protocol:slic
From 2014.igem.org
One-step Sequence and Ligation-Independent Cloning: Rapid and versatile cloning method for functional genomics studies
- Streak E.coli cells on an LB plate with or without antibiotics.
- Allow cells to grow at 37°C overnight.
- Place three or four colonies in 5 mL LB media (+ antibiotic selection if necessary), grow overnight at 37°C.
- Dilute the preculture to OD600 = 0,05 in 100 mL LB.
- Allow cells to grow at 37°C (250 rpm), until OD600= 0.4 (about 2-3 hours).
- Transfer cells to 2 centrifuge bottles (50 mL), and place cells on ice for 20 minutes.
- Centrifuge cells in Sorval GSA rotor at 4°C for 10 minutes at 5 000 rpm. Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room.
- Discard the supernatant and resuspend cells in 1/2 Vol of cold 50 mM CaCl2. Incubate on ice for 20 minutes.
- Centrifuge cells using Sorval RT6000B rotor at 4°C for 10 minutes at 5 000 rpm.
- Discard the supernatant and resuspend cells (by pipetting) in 1/20 Vol cold 50 mM CaCl2 containing 15% glycerol. Transfer 500 µL of cells into (1.5 mL) Ependorff tubes placed on ice. Cells stored at -80°C can be used for transformation for up to ~6 months.
- Add 2 µL of 0,5, 10 and 50 ng/L of Transformation Efficiency Kit DNA to 100 µl of competent cells.
- Incubate the mixture on ice for 35 minutes.
- Transfer the reaction to a 42°C water bath for exactly 2 minutes.
- Incubate on ice for 5 minutes.
- Add 900 µL of LB medium to each tube and incubate at 37°C for 1 hour to allow cells to recover and express the antibiotic resistance marker.
- Spread the appropriate quantity of cells (50 to 200 µl) on selective media. Store the remaining cells at 4°C.
- Incubate all plates overnight at 37°C (agar side up).
