RbCl Competent Cell Preparation
Solutions and Supplies
- sterilized 250 mL centrifuge bottles
- sterilized 1.5 mL microfuge tubes (at least 50)
- sterilized 100 mL LB in 250 mL flask
- filter sterilized 100 mL chilled RF1 (33 mL would be used)
- filter sterilized 50 mL chilled RF2 (4 mL would be used)
- 1 LB plate without any antibiotic and 6 LB plates with ampicillin (or other antibiotic) resistance
|Combine for 100 mL:
| Rubidium Chloride (RbCl, sigma R2252)||1.21 g||100 mM
| Manganese(II) chloride tetrahydrate (MnCl2·4H2O, sigma M3634)||0.99 g||50 mM
| Potassium acetate||0.294 g||30 mM
| Calcium chloride dihydrate (CaCl2·2H2O)||0.148 g||10 mM
| Glycerol||15 g or 12 mL||15% wt/vol
- Adjust final pH to 5.8 using 0.2 M acetic acid (maybe 400 μL for 33 mL). Filter-sterilize.
- Glacial acetic acid: 1.049 g·cm-3 / 60.05 g·mol-1 = 17.47 M
|Combine for 50 mL:
| MOPS (sigma M1254)||0.105 g||10 mM
| Rubidium Chloride (RbCl, sigma R2252)||0.06 g||10 mM
| Calcium chloride dihydrate (CaCl2·2H2O)||0.55 g||75 mM
| Glycerol||7.5 g or 6 mL||15% wt/vol
- Adjust final pH to 6.8 using 1 M NaOH (maybe 200 μL for 30 mL). Filter-sterilize.
Cell Preparation Procedure
- Streak DH5α from frozen glycerol stock on the LB plate.
- Incubate at 37 °C, over night.
- Prepare sterilized LB.
- Pick up a single colony from the LB plate.
- Inoculate to 3 mL sterilized LB.
- Incubate at 37 °C, over night.
- Put RF1, RF2, centrifuge tube and eppendorf tubes into the 4 °C refrigerator.
- Put RF1, RF2, centrifuge tube and eppendorf tubes on ice.
- Inoculate 1ml of over night culture to 100 mL of LB in flask.
- Monitor OD600 from initial until 0.2 to 0.6. [0.4 - 0.55 optimum]
- Transfer culture to centrifuge bottle and chill on ice 10 -15 min.
- Pellet cells by centrifugation at 2700 g (4200 rpm in an F14 6x250y rotor) for 10 min at 4 °C.
- 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 (NO VORTEX).
- Optimally, resuspend using a 25 mL disposable pipet (RbCl will permanently stain glass pipets).
- 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 580 g (1950 rpm in an F14 6x250y rotor) for 15 min at 4 °C.
- Decant liquid and gently resuspend in 1/25 original volume (4 mL) chilled RF2 buffer.
- Incubate cells/RF2 on ice for 15 min.
- 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 -80 °C.
Determine transformation efficiency
- Dilute control plasmid DNA (known DNA conc) to 1 ng/μL and transform using 1 μL.
- Thaw competent cells on ice.
- Compare previous lot to current lot .
- Combine 1 μL of diluted pDNA and 100 μL competent cells.
- Incubate on ice 30-60 min (40 min optimum).
- Heat shock at 42 °C for 90 sec, place on ice 5 - 15 min.
- Add 900ul LB and incubate at 37°C for 30-60 min (45 min optimum).
- Plate 100 μL onto antibiotic plate.
- Dilute the culture 10 times and plate 100 μL onto antibiotic plate.
- Dilute the culture 100 times and plate 100 μL onto antibiotic plate.
- Incubate all the plates at 37 °C, over night.
- Count the colonies in the next morning.