Heat-Shock Transformation of E. Coli

Notes

• Remember to double-check the required antibiotic for selection.
• This technique will work for most plasmids. Even quick and dirty mini-prep extracted plasmids.

Source

Standard lab protocol.

Materials

• Plasmid mini-prep
• TOP10 chemically competent E. coli cells (or your strain of choice)
• Petri-dishes plus appropriate antibiotics for selection (2 per each transformation reaction)
• Ice bath
• LB broth
• LB Agar
• Antibiotics for selection of common plasmids:
  • pCR8 – Spectinomycin
  • D-TOPO – Kanamycin

Equipment

Method

1. Liquefy LB Agar in the microwave, a couple of mins at 50% power. Place in a water bath at 65°C to keep liquid.
2. Defrost competent cells and plasmids on ice for approximately 10 mins
3. Add 1-2µl of the plasmid to competent cells, flick, incubate for 7 mins (anywhere between 2-30 mins) on ice.
4. Heat shock in 42°C bath for 90 seconds, put back onto ice.
5. Add LB broth to incubation tubes 700µl (anywhere between 400µl and 1000µl)
6. Incubate @ 37°C on Ratek shaker, 220 RPMs, for approximately 1hr (longer gives more various clones which isn’t a good thing).
7. Make up 2 plates for each transformation reaction:  For each petri dish: 
   1. Add approximately 20mL of LB agar to a falcon tube.
   2. Add 1µl of antibiotic stock for every 1000µl of LB agar.
   3. Pour Petri dishes, let sit to solidify for approximately 30 mins.
8. Plate out  30 or 60 µL of transformed cells onto 2 agar plates and spread.
9. Incubate overnight at 37°C.
10. Following day, select colonies and grow in LB broth containing the appropriate antibiotic for selection.

Use quick and dirty mini-prep protocol or {mini-prep extraction kit} to extract plasmids from overnight culture.