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.

Glycerol Stocks

1. Pick Single colonies from agar plates
2. Innoculate 5ml LB broth overnight.
3. Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.
4. Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.

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.

Miniprep

Miniprep using Thermo Scientific GeneJET Plasmid Miniprep Kit

All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g

1. Resuspend the pelleted cells in 250 ul of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
   Note. Ensure RNase A has been added to the Resuspension Solution.
2. Add 250 ul of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
   Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
   
3. Add 350 ul of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
   Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
   Note.Do not add bleach to the flow-through.
7. Add 500 ul of the Wash Solution (diluted with ethanol) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure (step 7) using 500 ul of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJETspin column into a fresh 1.5 ml microcentrifuge tube. Add 50 ul of the Elution Bufferto the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room tempera ture and centrifuge for 2 min.
    Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and in
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