Kafatos:competent cells

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Preparation of competent cells

  1. Grow 5 ml overnight of the strain in 2 X YT in a standard container. Screw cap tight and lie flat on the shaker for good aeration.
  2. Dilute overnight culture 1/100 - 1/200 to 25, 50 or 100 ml 2 X YT broth in flasks 5 - 10 X culture volume (i.e. 25 ml in 250 ml flask etc.).
  3. Grow to early log phase (OD600 = 0.2 - 0.4) (90 - 180 min depending on the strain)
  4. Collect cells by centrifugation (4000-5000 rpm for 5 min or 5000 x g for 5 min) at 4 C
  5. Keep the cells ice cold in all further steps.
  6. Resuspend the cells in 1/2 culture volume of 0.1 M ice-cold CaCl2. Hold on ice for minimum of 30 min, prefably 1 - 2 h.
  7. Collect cells as before and gently resuspend them in 1/10 culture volume 0.1 M CaCl2.
  8. Competent cells can be stored almost indefinitely by adding ice-cold sterile glycerol to a final concentration of 10% (v/v). Mix and leave on ice for 30 min, then store at -70 C

Transformation

  1. To transform, mix 0.1 ml aliquots of cells with DNA (1 - 10 ng). Leave on ice for 10 - 30 min.
  2. Heat shock at 42 C for 2 min (90 - 120 s is OK)
  3. Add 0.9 ml 2 X YT or LB and allow expression for 30 - 60 min before plating.
  4. If using X-gal and IPTG (optional), place 0.1 ml expression mix on the plate, add 40 µl X- gal and 4 µl IPTG, and spread together. This ensures that wherever cells go, X-gal does too!. Better still, add IPTG and X-gal to the plate (0.5 ml X-gal and 50ml IPTG per 100 ml agar).

Media, buffers and solutions

  1. YT,2x
  2. LB
  3. CaCl2 0.1M
  4. MgCl2, 0.1M
  5. glycerol
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