Wiese Lab:Competent Cell Prep

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Solutions and Supplies

 * sterilized 250 ml centrifuge bottles
 * sterilized 1.5 ml microfuge tubes
 * 300 ml LB in 2L flask
 * 100 ml LB in 1L flask
 * 100 ml chilled RF1 per 300 ml E. coli culture (or 33 ml per 100 ml culture)
 * 24 ml chilled RF2 per 300 ml E. coli culture (or8 ml per 100 ml culture)
 * Prepare RF1 and RF2 and store at 4oC.  Stable for > 1yr:

RF1

 * Adjust final pH to 5.8 using 0.2M acetic acid. Filter-sterilize.

RF2

 * Adjust final pH to 6.8 using NaOH. Filter-sterilize.

Prepare competent bacterial cells using RbCl2

 * - A 300-ml culture will yield ~ 60 aliquots of 400 ul competent cells
 * - A 100-ml culture will yield ~ 20 aliquots of 400 ul competent cells


 * Streak DH5alpha from frozen glycerol stock.
 * Prepare a 2 ml culture using a single CFU and 2 ml L Broth.
 * Shake at 37C O/N.

Next a.m.

 * Inoc 300 ml sterile LB in 2L flask using 300 ul of O/N culture.  (1:1000 dilution)
 * Monitor OD 550 from initial until 0.2 to 0.6. [0.4 to 0.55 optimum]
 * Transfer culture to centrifuge bottle and chill on ice 10 -15 min.
 * Pellet cells by centrifugation at 3000 rpm 12-15 min at 4C.
 * Decant liquid and resuspend in 1/3 original volume (100 ml or 33 ml) chilled RF1 buffer.
 * Optimally, resuspend using a 25-ml disposable pipet.
 * RbCl will permanently stain glass pipets.
 * Note: resuspend gently, DO NOT VORTEX.
 * Want to maintain pili structures on surface of E coli cell.
 * Continue mixing until cells are evenly resuspended and no clumps are visible.
 * Incubate cells/RbCl buffer 1 on ice for 45min to 60 min.
 * Pellet cells by centrifugation at 3000 rpm 12-15 min at 4C.
 * Decant liquid and gently resuspend in 1/12.5 original volume (24 ml or 8 ml) chilled RF2 buffer.
 * Incubate cells/RbCl buffer 2 on ice for 15min.
 * Distribute 400 ul into chilled 1.5 ml microfuge tubes and freeze on dry ice.
 * Store at -80C.

Determine transformation efficiency

 * Dilute control plasmid DNA (known DNA conc) to 2 ng/ul and transform using 1 ul.
 * Thaw competent cells on ice.
 * Compare previous lot to current lot
 * Combine 1ul of diluted pDNA and 200 ul competent cells.
 * Incubate on ice 30-60 min (40 min).
 * Heat shock at 42C for 1 min, place on ice 5 - 15 min.
 * Add 500 ul LB and incubate at 37C for 30-60 min (45 min).
 * Plate 10 ul and 100 ul onto antibiotic plate.