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 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 -80C.
- 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 1 ml LB and incubate at 37oC for 30-60 min (45 min).
- 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
