Transformation Of Ligated Dna Into Bacteria

Protocol 1. Preparation of Competent Cells for Transformation

Competent Cells for CaCl2 Transformation prior to Ligation

1. Prepare LB medium and LB plates. This should be done 2 to 3 days prior to ligation.

2. Streak an appropriate E. coli strain (DH5aF', Top 10 or JM109) directly from a small amount of frozen stock stored at -80°C onto the surface of

FIGURE 4.2 Streaking of bacterial stock in a zig-zag pattern on an LB plate. Well-isolated bacterial colonies will be generated 14 h later in the last streaking area in the plate.

an LB plate in a zig-zag pattern (Figure 4.2) using a sterile platinum wire loop. Invert the plate and incubate at 37°C for 12 to 16 h. Bacterial colonies will be formed.

Notes: It is not necessary to thaw frozen bacteria completely at room temperature or 0°C. A tiny bit of bacteria adhering to the wire loop is sufficient for inoculation.

3. Inoculate a well-isolated colony from the plate at step 2 into 5 ml of LB medium. Incubate at 37°C overnight with shaking at 160 rpm.

4. Add 0.5 ml of cells from step 3 to 100 ml of LB medium. Incubate at 37°C with shaking at 160 rpm. Four hours later, take 1 ml of the culture and measure the OD600 or A600 every 20 to 30 min until the A600 reaches 0.5 to 0.6. This usually takes 5 to 8 h.

Tip: Never allow bacteria to overgrow. If the density is higher than 0.65, the culture is too dense and transformation efficiency will be low.

5. Chill the cells on ice for 20 min and centrifuge at 5000 x g for 5 min at 4°C. Aspirate the medium.

6. Resuspend the cells in half of the original culture volume of sterile-filtered 0.1 M CaCl2 (50 ml) that has been prechilled on ice.

7. Incubate the cells on ice for 45 to 60 min.

8. Centrifuge at 5000 x g for 10 min at 4°C and gently resuspend the cells in 20 ml (1/20 original volume) of ice-cold 0.1 M CaCl2 solution.

9. Add sterile glycerol dropwise to the cell solution with gentle swirling to a final concentration of 15% (v/v). Aliquot the cells at 0.2 ml/tube, freeze and store at -80°C until use.

10. Take one aliquot to test efficiency of transformation.

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