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.
- 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
|
