15 Cloning Vectors
237
• Spectrophotometer
• Centrifuge
• Water bath
• Incubator
• Sterile toothpicks
Procedure
1. Prepare an overnight culture ofHB101: Add 10 ml of sterile LB medium ln advance
sterile to a culture flask. Transfer one colony of HB101 from the stock
plate to the culture flask. Place the culture tlask in a shakerat 3 7° C and let
the cells grow overnight.
Standardreaction
2. Add 45 ml ofLB medium and 0.5 ml of the HB 101 overnight culture to a
sterile culture flask and place it in the shaker at 37°C.
3. Grow the cells to an OD600 of0.3-0.5 (log-phase). This requires approximately 1.5-2 hours.
4. Incubate the cells for 15-30 minutes on ice.
5. Centrifuge the cells at 2500 x g for 5 min at 5°C.
6. Gently resuspend the pellet in 25 ml of ice-cold 50 mM CaCh. The cells
are now very fragile.
7. Place on ice for 15 min.
8. Centrifuge at 2500 x g for 5 min at 5°C.
9. Gently resuspend the pellet in 3.3 ml of ice-cold 50 mM CaCh. The cells
are now competent.
10. The competent cells can be stored overnight at 4°C. For long-term storage mix 600 J.ll of competent cells with 150 J.ll of sterile ice-cold glycerol
in an Eppendorf tube. Store at -70°C. Thaw on ice before use.
11. In a sterile Eppendorf tube mix 0.1-0.5 J.lg of plasmid or recombinant
plasmid DNA with 200 J.ll of competent cells.
Making cells
competent
Storage of
competent cells
Transformation
237
• Spectrophotometer
• Centrifuge
• Water bath
• Incubator
• Sterile toothpicks
Procedure
1. Prepare an overnight culture ofHB101: Add 10 ml of sterile LB medium ln advance
sterile to a culture flask. Transfer one colony of HB101 from the stock
plate to the culture flask. Place the culture tlask in a shakerat 3 7° C and let
the cells grow overnight.
Standardreaction
2. Add 45 ml ofLB medium and 0.5 ml of the HB 101 overnight culture to a
sterile culture flask and place it in the shaker at 37°C.
3. Grow the cells to an OD600 of0.3-0.5 (log-phase). This requires approximately 1.5-2 hours.
4. Incubate the cells for 15-30 minutes on ice.
5. Centrifuge the cells at 2500 x g for 5 min at 5°C.
6. Gently resuspend the pellet in 25 ml of ice-cold 50 mM CaCh. The cells
are now very fragile.
7. Place on ice for 15 min.
8. Centrifuge at 2500 x g for 5 min at 5°C.
9. Gently resuspend the pellet in 3.3 ml of ice-cold 50 mM CaCh. The cells
are now competent.
10. The competent cells can be stored overnight at 4°C. For long-term storage mix 600 J.ll of competent cells with 150 J.ll of sterile ice-cold glycerol
in an Eppendorf tube. Store at -70°C. Thaw on ice before use.
11. In a sterile Eppendorf tube mix 0.1-0.5 J.lg of plasmid or recombinant
plasmid DNA with 200 J.ll of competent cells.
Making cells
competent
Storage of
competent cells
Transformation