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pUC-T Easy Vector Systems INSTRUCTIONS FOR USE OF PRODUCTS T1101

Cloning PCR Products with pUC-T Easy Vectors

2. Briefly centrifuge the pUC-T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tube.

3. Set up ligation reactions as described below. Vortex the Solution I vigorously

Thaw competent cells on ice. Add 50μl cells to 2μl of the ligation reaction. Incubate on ice for

4. Mix the reactions by pipetting. Incubate the reactions 1 hour at room temperature. Alternatively, incubate the reactions overnight at 4°C for the maximum number of transformants.

Transformation of DH5-αor Other Strain High Efficiency Competent Cells

1. Prepare LB/ampicillin/IPTG/X-Gal plates.

2. Centrifuge the ligation reactions briefly. Add 2μl of each ligation reaction to a sterile 1.5ml tube on ice. Prepare a control tube with 0.1ng of uncut plasmid.

3. Place the DH5-αHigh Efficiency Competent Cells in an ice bath until just thawed (5 minutes). Mix cells by gently

4. Carefully transfer 50μl of cells to the ligation reaction tubes from Step 2. Use 100μl of cells for the uncut DNA control tube. Gently flick the tubes and Incubate on ice for 20 minutes.

5. Heat-shock the cells for 45–50 seconds in a water bath at exactly 42°C. DO NOT SHAKE. Immediately return the tubes to ice for 2 minutes.

6. Add 950μl room temperature SOC medium to the ligation reaction transformations