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

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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)