Test

RbCl Competent Cell Prep

Solutions and Supplies

• sterilized 250 ml centrifuge bottles
  
• sterilized 1.5 ml microfuge tubes (at least 50)
• sterilized 100 ml LB in 250ml flask
• filter sterilized 100 ml chilled RF1 per 300 ml E. coli culture (or 33 ml per 100 ml culture)
• filter sterilized 12 ml chilled RF2 per 300 ml E. coli culture (or 4 ml per 100 ml culture)

RF1

Combine for 1 L:		[final]
Rubidium Chloride (RbCl, sigma R2252)	12.1 g	100 mM
Manganese(II) chloride tetrahydrate (MnCl2 4H2O, sigma M3634)	9.9 g	50 mM
Potassium acetate	2.94g or 30 ml of 1M stock, pH 7.5	30 mM
Calcium chloride dihydrate (CaCl2 2H2O)	1.48 g	10 mM
Glycerol	150 g or 120ml	15% wt/vol

• Adjust final pH to 5.8 using 0.2M acetic acid (maybe 400ul for 33ml). Filter-sterilize.
• Glacial acetic acid: 1.049g cm-3 / 60.05g mol-1 = 17.47 M

RF2

Combine for 1 L:		[final]
MOPS (sigma M1254)	2.1g	10 mM
Rubidium Chloride (RbCl, sigma R2252)	1.2 g	10 mM
Calcium chloride dihydrate (CaCl2 2H2O)	11 g	75 mM
Glycerol	150 g or 120ml	15% wt/vol

• Adjust final pH to 6.8 using 1M NaOH (maybe 200ul for 30ml). Filter-sterilize.

Cell Preparation Procedure

Day 1

• Streak DH5alpha from frozen glycerol stock on the LB plate.
• Incubate at 37oC, over night.
• Prepare sterilized LB.

Day 2

• Pick up a single colony from the LB plate.
• Inoculate to 3ml sterilized LB.
• Incubate at 37oC, over night.
• Put RF1, RF2, centrifuge tube and eppendorf tubes into the 4oC refrigerator.

Day 3

• Put RF1, RF2, centrifuge tube and eppendorf tubes on ice.
• Inoculate 1ml of over night culture to 100ml of LB in flask.
• Monitor OD600 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 2700g (4200rpm in an F14 6x250y rotor) for 10 min at 4oC.
• Decant liquid and stand the bottle in an inverted position for < 1 min.
• resuspend in 1/3 original volume (33 ml) chilled RF1 buffer gently.
• 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/RF1 on ice for 15 min.
• Pellet cells by centrifugation at 580g (1950rpm in an F14 6x250y rotor) for 15 min at 4oC.
• Decant liquid and gently resuspend in 1/25 original volume (4 ml) chilled RF2 buffer.
• Incubate cells/RbCl buffer 2 on ice for 15min.
• Get eppendor tubes and box ready.
• Aliquot 100 ul each into chilled 1.5 ml eppendorf tubes and freeze on dry ice (or ice).
• Store at -80oC.

Determine transformation efficiency

• Dilute control plasmid DNA (known DNA conc) to 1 ng/ul and transform using 1 ul.
• Thaw competent cells on ice.
• Compare previous lot to current lot
• Combine 1ul of diluted pDNA and 100 ul competent cells.
• Incubate on ice 30-60 min (40 min optimum).
• Heat shock at 42oC for 90 sec, place on ice 5 - 15 min.
• Add 900ul LB and incubate at 37oC for 30-60 min (45 min optimum).
• Plate 100 ul onto antibiotic plate.
• Dilute the culture 10 times and plate 100 ul onto antibiotic plate.
• Dilute the culture 100 times and plate 100 ul onto antibiotic plate.
• Count the numbers of colonies in the next morning.