Monday, June 23, 2008

Competent Cells

CaCl2 METHOD

(All activities are done in aseptically in laminar flow cabinet, except for centrifuging).

  1. Prepare overnight culture – pick a colony of E. coli on agar plate and grow in 3 mL LB broth. Incubate at 37 oC overnight.
  2. Use 1 mL overnight culture to inoculate 100 mL LB broth in 500 mL erlenmeyer flask.
  3. Incubate at 37 oC with shaking (200 rpm).
  4. Grow to OD 600 = 0.4—0.5 (optimal for DH5α). Usually takes about 3 hours.
  5. Make 2 x 50 mL aliquots in oakridge tubes, centrifuge at 500 rpm for 15 minutes. Discard supernatant.
  6. Resuspend pellet in 10 mL ice cold CaCl2 solution.
  7. Centrifuge at 5000 rpm for 15 minutes, discard supernatant.
  8. Resuspend each pellet in 1 mL ice cold CaCl2 solution.
  9. Combine and make 200 µL aliquots in sterile 1.5 mL microcentrifuge tubes.
  10. Snap freeze on dry ice/ETOH.
  11. Store at -70 oC, labelled CC.

For transformation (heat-shock method):

  1. Mix 100—200 µL of CC with 1 µL plasmid DNA (or 10 µL ligation mixture) in a sterile 1.5 mL microcentrifuge tube. Incubate on ice (or put in freezer) for 30 minutes.
  2. Heat shock treatment: put tube in dry bath or water bath at 42 oC for 2 minutes.
  3. Immediately put on ice for 10 minutes.
  4. Cell/DNA mixture is spread on agar plate (plus ampicillin or other antibiotic). Incubate at 37 oC overnight.

TSS METHOD (1)

  1. Inoculate 3 mL of LB broth with an E. coli colony from agar plate. Incubate at 37 oC overnight, with shaking.
  2. Dilute 1:100 fresh overnight culture of bacteria into pre warmed LB broth and incubate cells with shaking (225 rpm) to an OD 600 of 0.3—0.4 (approximately 3 hours).
  3. Add an equal volume of ice-cold 2X TSS and mix gently. [TSS is LB broth with 10% PEG (MW 3350-8000), 5% DMSO, and 20-50 mM Mg 2+ (MgSO4 or MgCl2) at a final pH of 6.5].
  4. For long term storage, cells are frozen immediately in a dry ice/ethanol bath and stored at -70 oC.

TSS METHOD (2)

  1. Inoculate 50 mL LB broth with 0.5 mL fresh overnight culture and incubate at 37 oC with shaking until OD 600 reaches 0.3-0.4.
  2. Spin to pellet cells after mixing (5000 rpm, 3 minutes) and add 2 mL of 1 X TSS.

For transformation (1):

  1. A 0.1 mL aliquot of competent cells pipetted into a cold polypropylene tube containing 1 µL (100 pg) of plasmid DNA or ligation mixture, and cell/DNA suspension is mixed gently. [When frozen cells are used, cells are thawed slowly on ice and used immediately].
  2. The cell/DNA mixture is incubated for 5—60 minutes at 4 oC.
  3. A 0.9 mL aliquot of TSS (or LB broth) plus 20 mM glucose is added, and cells are incubated at 37 oC with shaking (225 rpm) for 1 hour to allow expression of the antibiotic resistance gene.
  4. Transformants are selected with standard methods.