Team:Aix-Marseille/Protocol:slic

From 2014.igem.org

(Difference between revisions)

Latest revision as of 10:50, 11 August 2014

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.

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-             Streak <i>E.coli</i> cells on an LB plate with or without antibiotics.
+             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).
            </li>
            <li>
-             Allow cells to grow at 37°C overnight.
+             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).
            </li>
            <li>
-             Place three or four colonies in 5 mL LB media (+ antibiotic selection if necessary), grow overnight at 37°C.
+             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 H<sub>2</sub>O up to 10 μL.
            </li>
            <li>
-             Dilute the preculture to OD600 = 0,05 in 100 mL LB.
+             Add 0.5 μL of T4 DNA polymerase (3 U/μL NEB) to the mixture and incubate at room temperature for 2 minutes.
            </li>
            <li>
-             Allow cells to grow at 37°C (250 rpm), until OD600= 0.4 (about 2-3 hours).
+             Put the reaction mixture on ice immediately to stop the reaction and incubate on ice for 10 minutes.
            </li>
            <li>
-             Transfer cells to 2 centrifuge bottles (50 mL), and place cells on ice for 20 minutes.
+             Thaw chemically competent <i>E.coli</i> cells on ice for 10 minutes.
            </li>
            <li>
-             Centrifuge cells in Sorval GSA rotor at 4°C for 10 minutes at 5 000 rpm.
+             Mix 90 μL of cells with 5 μL of the reactant.
            </li>
-          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.
            <li>
-             Discard the supernatant and resuspend cells in 1/2 Vol of cold 50 mM CaCl<sub>2</sub>. Incubate on ice for 20 minutes.
+             Incubate cells on ice for 20 minutes.
            </li>
            <li>
-             Centrifuge cells using Sorval RT6000B rotor at 4°C for 10 minutes at 5 000 rpm.
+             Heat shock cells at 42°C for 45 seconds.
            </li>
            <li>
-             Discard the supernatant and resuspend cells (by pipetting) in 1/20 Vol cold 50 mM CaCl<sub>2</sub> 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.
+             Incubate cells on ice for 2 minutes.
            </li>
            <li>
-             Add 2 µL of 0,5, 10 and 50 ng/L of Transformation Efficiency Kit DNA to 100 µl of competent cells.
+             Add 900 μL of LB broth to 90 μL of cells.
            </li>
            <li>
-             Incubate the mixture on ice for 35 minutes.
+             Incubate cells at 37°C for 1 hour.
            </li>
            <li>
-             Transfer the reaction to a 42°C water bath for exactly 2 minutes.
+             Plate cells on agar plates containing suitable antibiotics.
            </li>
            <li>
-             Incubate on ice for 5 minutes.
+             Incubate the plates at 37°C for 16 hour and analyze the colonies.
-          </li>
-          <li>
-            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.
-          </li>
-          <li>
-            Spread the appropriate quantity of cells (50 to 200 µl) on selective media. Store the remaining cells at 4°C.
-          </li>
-          <li>
-            Incubate all plates overnight at 37°C (agar side up).
            </li>
          </ol>

Team:Aix-Marseille/Protocol:slic

From 2014.igem.org

Latest revision as of 10:50, 11 August 2014

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