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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
- Digest vector with restriction enzyme(s) overnight, and purify the linearized vetor with a commercial PCR purification kit. Elute the DNA with elution buffer or 10 mM TrisCl, pH 8.0-8.5. Do not elute the DNA with water or TE. Measure the concentration of the vector with the Nanodrop (be careful and make the blank with 10 mM TrisCl, pH 8.0-8.5).
- Amplify your gene of interest by PCR using primers with ≥ 15 mer homology extension to the linearized vector end. We usually use 15 bp homology for single fragment cloning, and 20 bp homology for multiple fragment cloning. Purify the linearized vector with a commercial PCR purification kit. Elute the DNA with elution buffer or 10 mM TrisCl, pH 8.0-8.5. Do not elute the DNA with water or TE. Measure the concentration of the vector with the Nanodrop (be careful and make the blank with 10 mM TrisCl, pH 8.0-8.5).
- Mix the linearized vector and insert at a molar ratio of 1:2 in a 1.5 mL tube. Vector to insert molar ratio of 1:1 to 1:7 works well, but usually is used 1:2 for single fragment cloning, 1:2:2 for multiple fragments cloning. Add to the mixture 1 μL of 10X BSA, 1 μL of 10X NEB Buffer 2 and H2O up to 10 μL.
- Add 0.5 μL of T4 DNA polymerase (3 U/μL NEB) to the mixture and incubate at room temperature for 2 minutes.
- Put the reaction mixture on ice immediately to stop the reaction and incubate on ice for 10 minutes.
- Thaw chemically competent E.coli cells on ice for 10 minutes.
- Mix 90 μL of cells with 5 μL of the reactant.
- Incubate cells on ice for 20 minutes.
- Heat shock cells at 42°C for 45 seconds.
- Incubate cells on ice for 2 minutes.
- Add 900 μL of LB broth to 90 μL of cells.
- Incubate cells at 37°C for 1 hour.
- Plate cells on agar plates containing suitable antibiotics.
- Incubate the plates at 37°C for 16 hour and analyze the colonies.
