CaCl2 Competent Cells

This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.

Note: Never vortex competent cells. Resuspend by pipetting with large Pasteur pipettes.

1. The night before, inoculate a 5 ml culture and grow overnight with selection.
2. The day of the experiment dilute cells ~ 1:200 into selective media.
   For this example add 250 ul to 50 ml of selective media.
   Note: The protocol is easily scaled to increase the number of cells.
3. Grow the cells to an OD600 of 0.6 – 0.7.
   Use a large flask, 500ml, for good aeration.
   Use a baffled flask for fastest growth.
   This takes about 3 hours depending on the cells.
   Medium-heavy cloudiness by eye is fine.
4. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
5. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
6. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
7. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
8. Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
   Note: Frozen cells are only good once.Do not refreeze cells once thawed.

Electroporation

Preparation of Electrocompetent Cells

Competent cells should never be vortexted, as this will cause them to lyse and release salts into the media. Resuspend cells by pipeting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.

1. The night before the transformation, start an overnight culture of cells.
   5 ml LB Amp.
2. The day of the transformation, dilute the cells 100X. 100 ml LB Amp.
   Grow at 30°C for about 90 minutes.
3. Harvest the cells.
   When the cells reach an OD600 of between 0.6 and 0.8.
   Split the culture into 2x 50 ml falcon tubes, on ice.
   Centrifuge at 4 °C for 10 min at 4000 rpm.
4. Wash and combine the cells.
   Remove the supernatant.
   Resuspend the cells in 2x 25 ml of ice cold water.
   Combine the volumes in a single 50 ml falcon tube.
5. Wash the cells 2 more times.
   Centrifuge at 4 °C for 10 min at 4000 rpm.
   Resuspend in 50 ml of ice cold water.
   Repeat.
6. Wash and concentrate the cells for electroporation.
   Centrifuge at 4 °C for 10 min at 4000 rpm.
   Resuspend in 1-2 ml of ice cold water.
   We will use 200 ul of washed cells per transformation.

Dialysis of PCR or Digestion Products

DNA for electroporation must be free of salts to avoid arcing.

1. Float a filter in a Petri dish filled with water.
   Millipore membrane filter 0.025 uM.
   
2. Pipet one drop of PCR product onto the filter.
   200 ng is needed per transformation.
   20 - 100 ul fits well on one filter.
   
3. Collect the drop after 30 - 45 minutes.
   The volume will change, but the DNA is not lost.

Titre

Text

Note

1. Bla
2. BlaBla

Titre

Text

Note

1. Bla
2. BlaBla

Titre

Text

Note

1. Bla
2. BlaBla

Titre

Text

Note

1. Bla
2. BlaBla

Titre

Text

Note

1. Bla
2. BlaBla