IGEM:PennState/2006/PreparingChemicallyCompetentCells

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Contents

Making Cells Competent

Time: O’N then 3 hr next day

Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 "Trans. Using CaCl<sub>2</sub>"

Makes 64x50μL aliquots

Media/Reagents

  • LB
  • Cells (from plate)
  • CaCl2 Solution
  • Dry Ice (2nd floor Althouse or S. Frear)

Pre

  • Prepare CaCl2 solution1
  • Autoclave centrifuge bottles/tubes and chill on ice
  • Before centrifuge steps, make sure centrifuge is on and at 0°C

Competent Cells: Day 0

  1. Streak cells on LB plate
  2. Grow overnight at 37°C
Streaked plate with bacteria
Streaked plate with bacteria









Competent Cells: Day 1 (optional)

  1. Inoculate overnight starter culture (2 mL) w/ colony from plate

Competent Cells: Day 2

  1. Inoculate 80 mL2 with appropriate amount of O’N to obtain OD6000~0.1. Place cells on ice at OD600=0.375 (do not allow growth past an OD of 0.4, as this decreases subsequent transformation efficiency). Typically this will take 40-60 min (check every 5 min when OD is getting near 0.375).
  2. Ice for 10 min.
  3. Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
  4. Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
  5. Spin 1100g/5 min/0°C.
  6. Gently resuspend pellet in 8 mL ice-cold CaCl2. Let stand on ice for 30 min.
  7. Spin 1100g/5 min/0°C. Decant supernatant & resuspend well in 1.6 mL ice-cold CaCl2. Cells may stand on ice for 12-24 hrs to increase competency.
  8. Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs. Immerse immediately in crushed dry ice.
  9. Transfer to box(es) and store in -80°C freezer.



1CaCl2(per L)

  • 20 g bactotryptone
  • 5 g bacto-yeast extract
  • 0.5 g NaCl
  • 0.19 g KCl
  • Adjust to pH 7.0 w/ NaOH
  • Autoclave
  • Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)


2Procedure can be scaled up or down as necessary

Cloning

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