Plasmid Transformations
 Plasmids are circular DNA fragments that are contain DNA that is not necessary for the bacteria's survival. Plasmid transformations are a way to give cells abilities they would not normally have, such as antibiotic resistance.

For transformations, it is helpful to give the bacteria antibiotic resistance in addition to the desired gene. This procedure is written for an E. coli transformation, but of any bacteria may be used.

Materials

Ice

100mM CaCl2, sterile

Broth medium

Agar plates

Agar with appropriate antibiotics

Plasmid

Nuclease-free water

SOC media

Creating Chemically Competent Cells using CaCl2

If the bacteria you are working with is naturally competent, you may skip this step.

  1. Inoculate 10 mL of sterile LB with your desired E. coli strain. Culture overnight at 37 °C with shaking at 200 rpm.

  2. Re-inoculate 1 mL of the overnight culture into 99 mL of sterile LB and culture at 37 °C with shaking at 200 rpm until an Optical Density at 600 nm (OD600) of 0.3 – 0.5 (mid log phase) is reached.

  3. Split the culture between sterile centrifuge tubes, adding 1mL to each tube. Collect the cells by centrifugation for 5 minutes at ~ 7000 rpm. Discard the supernatant and use a P200 pipette to remove any drops that remain.

  4. Add 1mL of sterile 100mM CaCl2 to each pellet and gently resuspend. Allow the resuspended cells to chill on ice for 15 minutes.

  5. Collect the washed cells by centrifugation for 10 minutes at ~ 7000 rpm and discard the supernatant.

Cells are now ready to be transformed, but if you are storing competent cells, follow steps 6-9:

  1. Add 5 mL of sterile, ice-cold 100 mM CaCl2 supplemented with 15 % v/v glycerol to each cell pellet and gently resuspend them.

  2. Divide the resuspended cells into 50 μL aliquots in sterile, ice-cold Eppendorf tubes.

  3. Store the chemically competent cells at -80 °C. They should remain competent for at least 1 year.

  4. Quantify the transformation efficiency of your chemically competent cells by transforming them with a known amount (say 1 µL of 10 – 50 ng/µL) of plasmid that contains a positive selection marker and count the number of transformant colonies.

Transforming Competent Cells

  1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).

  2. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.

  3. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.

  4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.

  5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells you are using).

  6. Put the tubes back on ice for 2 min.

  7. Add 250-1,000 μl SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.

*Pro-Tip* This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances.

  1. 50uL of the transformation onto an LB agar plate containing the appropriate antibiotic. If you have more than 50uL, plate the rest on a second LB agar plate containing the appropriate antibiotic.

*Pro-Tip* If the culture volume is too big, gently collect the cells by centrifugation and resuspend in a smaller volume of LB so that there isn't too much liquid media on the agar plates. If the agar plate doesn't dry adequately before the cells begin dividing, the bacteria diffuse through the liquid and won't grow in colonies.

  1. Incubate plates at 37°C overnight.