Competent Cells
Preparation of Bacillus subtilis competent cells
- Streak out the strain to be made competent on an LB agar plate as a large patch and incubate overnight at 30°C
- 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.
- Incubate the culture at 37°C with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.
- 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
- After 90 min incubation, pellet the cells by centrifugation (8,000 g, 5min) at room temperature.
- Carefully decant the supernatant into a sterile container and save.
- Gently resuspended the cell pellet in 18 ml of the saved supernatant and add 2 ml of sterile glycerol; mix gently
- 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.
Transformation
Cells Preparation :
- Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath
- Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently
- 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.
- Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.
Digestion :
- 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.
- Incubate tube at appropriate temperature (usually 37°C) for 1 hour.
- Always follow the manufacturer’s instructions.
- To visualize the results of your digest, conduct gel electrophoresis
Vector Preparation :
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).
Deletion
Deletion Step 1 :
Deletion Step 2 :
Deletion Step 3 :
Other
Whatever Step 1 :
Whatever Step 2 :
Un autre truc Step 1 :
Un autre truc Step 2 :
