1.Streak out the strain to be made competent on an LB agar plate as a large patch and incubate overnight at 30°C

2. The following morning scrape the cell growth off the plate and use to inoculate fresh, pre-warmed, SpC medium (20 ml) to give an OD600 reading of about 0.5.

3. Incubate the culture at 37°C with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.

4. When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 min) inoculate 200 ml of pre-warmed, SpII medium with 2 ml of stationary-phase culture and continue incubation at 37°C with slower aeration

5. After 90 min incubation, pellet the cells by centrifugation (8,000 g, 5min) at room temperature.

6. Carefully decant the supernatant into a sterile container and save.

7. Gently resuspended the cell pellet in 18 ml of the saved supernatant and add 2 ml of sterile glycerol; mix gently

8. Aliquot the competent cell (0.5 ml) in sterile tubes, freeze rapidly in liquid nitrogen or a dry-iced/ethanol bath or ice/isopropanol bath and store -70.

1. Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath

2. Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently

3. In a sterile test tube add competent cell (0.2~0.5 ml) to the DNA solution (<0.1 ml) and incubate in a roller drum at 37.

4. Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.

1. Select restriction enzymes to digest your plasmid. Determine an appropriate reaction buffer by reading the instructions for your enzyme. In a 1.5mL tube combine the following:
- DNA
- Restriction Enzyme(s)
- Buffer
- dH2O up to total volume

Mix gently by pipetting.

2. Incubate tube at appropriate temperature (usually 37°C) for 1 hour.

3. Always follow the manufacturer’s instructions.

4. To visualize the results of your digest, conduct gel electrophoresis

Combine the following in a PCR or Eppendorf tube:

- 25ng Vector DNA

- 75ng Insert DNA

- Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)

- 0.5-1μL T4 DNA Ligase

- H20 to a total of 10μL

Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).

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