IGEM:PennState/2006/PreparingChemicallyCompetentCells

=Making Cells Competent= Time: O’N then 3 hr next day

''Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 "Trans. Using CaCl2" ''

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



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 OD600~0.1. 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.

1TB Buffer (CaCl2)(per L)
 * 10mM pipes
 * 15 mM CaCl2
 * 250 mM KCl
 * note: these will not dissolve until pH is adjusted
 * Adjust pH to 6.7 with KOH
 * Add MnCl2 for final Conc. of 55mM
 * Filter Sterilize
 * Store at 4°C

2SOB
 * 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)

3Procedure can be scaled up or down as necessary

Cloning

Main

Preparation of ultra-competent E. coli cells for transformation Based on Inoue et al (1990), Gene, 96:23-28, with modifications.

1. Culture cells (DH5a in my case) on LB agar plate at 37oC overnight. 2. Pick up 10 -12 large colonies and culture in 250ml SOB in a 1L flask, 19oC with vigorous shaking to OD600=0.5 (normally it takes 24-36hrs) 3. Place the flask in ice for 10 min. 4. Pelleting the cell by spining at 4000rpm for 10 min at 4oC. 5. Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.  6. Spin at 4000rpm for 10 min at 4oC 7. Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO (the DMSO needs to be stored at -20oC o/n before use). 8. Aliquote the cell to 50 to 500ul for transformation or store at -70oC

* Note: The E. coli cells prepared this way are normally 100 to 1000 times more efficient than normal calcium method, so do not plate too dense!

1. SOB solution:

o 0.5% yeast extract o 2% tryptone o 10mM NaCl o 2.5mM KCl o 10mM MgCl2 o 10mM MgSO4 o Dissolve in nanopure water and autoclave to sterilize.

* TB solution:

o 10mM PIPES o 15mM CaCl2 o 250mM KCl o Dissolve in nanopure water and adjust pH to 6.7 with KOH or HCl and then add MnCl2 to 55mM, and adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4oC

Comments or Questions? Send to me at:chunming.liu@wur.nl     http://www.hybtech.org/Liu/uccell.html