RbCl competent cell: Difference between revisions
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===RF1=== | ===RF1=== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Combine for | | align="center" style="background:#f0f0f0;"|'''Combine for 100 mL:''' | ||
| align="center" style="background:#f0f0f0;"| | | align="center" style="background:#f0f0f0;"| | ||
| align="center" style="background:#f0f0f0;"|'''[final]''' | | align="center" style="background:#f0f0f0;"|'''[final]''' | ||
|- | |- | ||
| Rubidium Chloride (RbCl, sigma R2252)|| | | Rubidium Chloride (RbCl, sigma R2252)||1.21 g||100 mM | ||
|- | |- | ||
| Manganese(II) chloride tetrahydrate (MnCl<sub>2</sub>·4H<sub>2</sub>O, sigma M3634)|| | | Manganese(II) chloride tetrahydrate (MnCl<sub>2</sub>·4H<sub>2</sub>O, sigma M3634)||0.99 g||50 mM | ||
|- | |- | ||
| Potassium acetate|| | | Potassium acetate||0.294 g or 3 mL of 1M stock, pH 7.5||30 mM | ||
|- | |- | ||
| Calcium chloride dihydrate (CaCl<sub>2</sub>·2H<sub>2</sub>O)|| | | Calcium chloride dihydrate (CaCl<sub>2</sub>·2H<sub>2</sub>O)||0.148 g||10 mM | ||
|- | |- | ||
| Glycerol|| | | 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. | *Adjust final pH to 5.8 using 0.2 M acetic acid (maybe 400 μL for 33 mL). Filter-sterilize. |
Revision as of 12:24, 25 September 2011
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 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 100 mL: | [final] | |
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 or 3 mL of 1M stock, pH 7.5 | 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
RF2
Combine for 1 L: | [final] | |
MOPS (sigma M1254) | 2.1 g | 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 120 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
Day 1
- Streak DH5α from frozen glycerol stock on the LB plate.
- Incubate at 37 °C, over night.
- Prepare sterilized LB.
Day 2
- 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.
Day 3
- 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.
- 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 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.