Wiese Lab:Competent Cell Prep

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RbCl Competent Cell Prep

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

Combine for 1 L:		[final]
RbCl	12 g	100 mM
MnCl2 4H2O	9.9 g	50 mM
K acetate	30 ml of 1M stock, pH 7.5	35 mM
CaCl2 2H2O	1.5 g	10 mM
Glycerol	150 g	15% wt/vol

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

RF2

Combine for 1 L:		[final]
MOPS		10 mM
RbCl	1.2 g	10 mM
CaCl2 2H2O	11 g	75 mM
Glycerol	150 g	15% wt/vol

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

Cell Preparation Procedure

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.

Calculate transformation efficiency	For example
conc pDNA	2 ng/ul
vol used	1 ul
total amt pDNA	2 ng