Competent cells

Protocol for making competent cells

Need competent cells for students in week 3 - make them in week 1 or 2.

0.5 M PIPES disodium salt(pH 6.7) (Alfa Aesar 3p B21835-22) or Fisher AC21509-1000

  • 17.3 g PIPES (disodium salt) in 80 mL H2O
  • OR 15.1 g PIPES in 80 mL H2O
  • initial pH = ~5.5
  • KOH pellets till it goes clear (checking pH!!)
  • final pH to 6.7 with 5M KOH
  • water to 100 mL
  • filter sterilize
  • freeze in 10 mL aliquots (enough for 500 mL transformation buffer)

Transformation buffer

Reagent cf (mM) per L .
MnCl2 55 10.88 g
CaCl2 15 2.2 g
KCl 250 18.65 g
PIPES (pH 6.7, 0.5M) 10 20 mL

filter sterilize. refrigerate


SOB Medium

Per Liter:

  • 950 ml of deionized H2O
    • 20 g Tryptone
    • 5 g Yeast Extract
    • 0.5 g NaCl
  • stir to dissolve
    • 2.5 mL 1M KCl (10 mL 250 mM KCl)
  • pH to 7 with NaOH
  • volume to 1 L
  • autoclave 20 min
  • cool
    • 10 mL sterile 1M MgCl (5 mL sterile MgCl2 2M)
    • ? 10 mL sterile 1M MgSO4?

SOB AGAR

15 g agar/L SOB

when cooled, add

20 mL 1 M MgSO4

12.5 µg/mL tetracycline (=0.83 mL 15 mg/mL tet per L)

or

50 µg/mL ampicillin as needed (= 1mL 50 mg/mL amp stock per L)