Haynes:TransformationPlasmids

Transformation of E. coli With Plasmid DNA

by Karmella Haynes, 2012

Chemically competent cells + plasmid miniprep 30 min.

1. Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
2. Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per ligation.
3. Dilute 0.5 μL plasmid DNA (concentration not important) in 10 μL sterile dH₂O in sterile 0.5 mL tubes.
4. Make a "negative control" sample by simply putting 10 μL sterile dH₂O in a tube without DNA.
5. Add 30 μL thawed cells each tube of diluted DNA (and the negative control). Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp or Kan resistance)
6. Label the pre-warmed plates with the antibiotic name, strain name, DNA name, your initials, and the date.
7. Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
8. Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.

Chemically competent cells + iGEM Registry sample 30 min.

1. Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
2. Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per ligation.
3. Locate the desired well in the Registry plate. Inject 10 μL sterile dH₂O into the well and pipette up and down to resuspend the dried DNA and indicator dye. Transfer each DNA solution to a sterile 0.5 mL tube.
4. Make a "negative control" sample by simply putting 10 μL sterile dH₂O in a tube without DNA.
5. Add 30 μL thawed cells each tube of diluted DNA (and the negative control). Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp or Kan resistance)
6. Label the pre-warmed plates with the antibiotic name, strain name, DNA name, your initials, and the date.
7. Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
8. Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.