Haynes:TransformationPlasmids

<- Back to Protocols

Traditional Method: Chemically competent cells + plasmid DNA

This method is useful for plasmids that carry cholramphenicol and zeomycin resistance.

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 chemically competent cells on ice just until thawed. You will need 30 μL cells per plasmid.
3. Prepare the DNA (two options)
   1. Plasmid Miniprep: Dilute 0.5 μL plasmid DNA (concentration not important) in 10 μL sterile dH₂O in sterile, labeled 0.5 mL tubes.
   2. iGEM Registry Sample: 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, labeled 0.5 mL tube.
4. Make a "negative control" sample by simply putting 10 μL sterile dH₂O in an 0.5 mL tube without DNA.
5. Add 30 μL thawed cells to 1 sterile, labeled 2.0 mL tube. Do this once for each different plasmid, and once for the negative control.
6. Transfer the plasmid DNA from the 0.5 mL tubes to the cells in each 2.0 mL tube. Tap the 2.0 mL tube to gently to mix the cells and DNA.
7. Incubate on ice for 10 min.
8. Heat shock: Transfer the tubes to 42°C for exactly 45 seconds (heat shock) on a heat block or water bath, then immediately place the tubes on ice for 1 minute.
9. Add 750 μL sterile SOC medium to each sample.
10. Close the caps tightly. Place the tubes in the shaking incubator, secured in a sideways position with lab tape. Incubate the tubes, with shaking, at 37°C for 45 minutes.
11. Pre-warm the agar plates: Incubate the selection agar plates (one per sample) at 37°C during the 45 minute recovery period.
12. Pellet the cells by centrifugation at top speed for 3 minutes at room temperature.
13. Discard the SOC supernatant.
14. Resuspend the pellet in 100 μL LB medium (plus proper antibiotic).
15. Pipette the total volume of cells onto the agar; spread using sterile glass beads.
16. Incubate the inverted plate(s) overnight at 37°C to get colonies.

Note: To store the colonies long term, seal the plate with parafilm and keep the plate at 4°C (inverted).

Traditional Method: Chemically competent cells + ligation

This method is useful for increasing the efficiency of plasmid up-take.

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 chemically competent cells on ice just until thawed. You will need 30 μL cells per ligation.
3. Add 30 μL thawed cells to to 1 sterile 2.0 mL tube (per ligation).
4. Add the total ligation reaction to the cells in each 2.0 mL tube. Pipette up and down gently to mix the cells and DNA.
5. Incubate on ice for 10 min.
6. Heat shock: Transfer the tubes to 42°C for exactly 45 seconds (heat shock) on a heat block or water bath, then immediately place the tubes on ice for 1 minute.
7. Add 750 μL sterile SOC medium to each sample.
8. Recovery: Close the caps tightly. Place the tubes in the shaking incubator, secured in a sideways position with tape. Incubate the tubes, with shaking, at 37°C for 45 minutes.
9. Pre-warm the agar plates: Incuabte the selection agar plates (one per sample) at 37°C during the 45 minute recovery period.
10. Pellet the cells by centrifugation at top speed for 3 minutes.
11. Discard the SOC supernatant.
12. Resuspend the pellet in 100 μL LB medium (plus proper antibiotic).
13. Pipette the total volume of cells onto the agar; spread using sterile glass beads.
14. Incubate the inverted plate(s) overnight at 37°C to get colonies.

Note: To store the colonies long term, seal the plate with parafilm and keep the plate at 4°C (inverted).

Quick Method: Chemically competent cells + plasmid DNA

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 plasmid sample.
3. Prepare the DNA (two options)
   1. Plasmid Miniprep: Dilute 0.5 μL plasmid DNA (concentration not important) in 10 μL sterile dH₂O in sterile 0.5 mL tubes.
   2. iGEM Registry Sample: 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 to 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.
Note 2: This method can be used for ligations, where construction is very straight-forward and your DNA material is highly concentrated. It works well for "forced" ligations (sticky overhangs, no unwanted annealing)