Lidstrom:Competent Cell Preparation: Difference between revisions
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====Recipes:==== | ====Recipes:==== | ||
== SOB-Mg growth medium: 1L == | |||
*CCMB | |||
{|border="2" cellpadding="1" | |||
|+Setting cell widths | |||
!width="120"|Ingredient | |||
!width="40"|Amount | |||
|- | |||
|Bacto Tryptone||20g | |||
| | |||
|- | |||
|Bacto Yeast Extract||5g | |||
| | |||
|- | |||
|1M NaCl||10mL | |||
| | |||
|- | |||
|1M KCl||2.5mL | |||
|} | |||
* Add water to make 1L | |||
* Autoclave | |||
* Dispense into smaller bottles for lower contamination risk | |||
== CCMB (1L) == | |||
{|border="2" cellpadding="1" | |||
|+Setting cell widths | |||
!width="180"|Ingredient | |||
!width="60"|Amount | |||
!width="140"|Final Concentration | |||
|- | |||
|Potassium Acetate, 1M, pH 7||10 mL||10mM | |||
| | |||
|- | |||
|Glycerol||100g||10% (w/v) | |||
| | |||
|- | |||
|CaCl2.2H2O||11.8g||80mM | |||
| | |||
|- | |||
|MnCl2.4H2O||4g||20mM | |||
| | |||
|- | |||
|MgCl2.6H2O||2.5mL||10mM | |||
|} | |||
# Prepare a 1M solution of potassium acetate, pH 7.0 using KOH. | |||
## Filter through a 0.2 uM membrane & store frozen | |||
# Prepare a solution of 10% potassium acetate, 10% glycerol | |||
# Add salts, allowing each to enter solution before adding the next. | |||
# Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base. | |||
# Filter through a 0.2 uM filter & store at 4<sup>o</sup>C. | |||
===Nicole/Andrew protocol for Chemically Competent cells=== | ===Nicole/Andrew protocol for Chemically Competent cells=== |
Revision as of 12:30, 1 March 2013
Back to Protocols
Chemically Competent E. Coli
Notes:
- You need fresh cells.
- Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.
- Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest.
- You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.
Amanda/Janet Protocol for Chemically Competent Cells
Supplies needed:
- Plate of E. Coli colonies
- SOB-Mg growth medium
- Sterilized 500 mL Erlenmeyer flasks
- 50 mL screw-cap polypropylene tubes
- Freezer tubes (1.5 mL)
- SOC medium for recovery after heat shock (LB/TB is fine instead)
Method
- Pick several colonies off a freshly streaked plate into ~1 mL SOB-Mg growth medium
- Grow cells overnight or several hours in media with appropriate antibiotics if available.
- Use more inoculum in the next step if cultures weren't grown overnight.
- Inoculate 50 mL SOB-Mg growth medium with this culture. Use 500 uL of stationary phase culture for 50 mL SOB-Mg medium.
- Incubate at 275 rpm, 37oC until OD600 is about 0.3, which corresponds to ~ 5*107cells/mL
- Higher OD isn't usually a problem for routine work.
- Collect in sterile 50 mL polypropylene centrifuge tube(s) and chill on ice for 10 minutes
- Pellet the cells at 750 - 1,000g (2500 rpm) for 14 min at 4oC. Decant the supernatant and invert tubes to remove excess culture medium.
- Disperse cells in ~1/3 volume of CCMB by gentle vortexing or rapping of the centrifuge tube.
- Incubate on ice for 20 minutes
- Centrifuge at 2500 rpm for 10 min at 4oC
- Resuspend cells in CCMB at 1/12 the original culture volume
- Make aliquots in eppendorf tubes, ideally on ice
- Flash freeze with liquid nitrogen
- Store at -80oC to preserve them for many months
Recipes:
SOB-Mg growth medium: 1L
Ingredient | Amount | |
---|---|---|
Bacto Tryptone | 20g | |
Bacto Yeast Extract | 5g | |
1M NaCl | 10mL | |
1M KCl | 2.5mL |
- Add water to make 1L
- Autoclave
- Dispense into smaller bottles for lower contamination risk
CCMB (1L)
Ingredient | Amount | Final Concentration | |
---|---|---|---|
Potassium Acetate, 1M, pH 7 | 10 mL | 10mM | |
Glycerol | 100g | 10% (w/v) | |
CaCl2.2H2O | 11.8g | 80mM | |
MnCl2.4H2O | 4g | 20mM | |
MgCl2.6H2O | 2.5mL | 10mM |
- Prepare a 1M solution of potassium acetate, pH 7.0 using KOH.
- Filter through a 0.2 uM membrane & store frozen
- Prepare a solution of 10% potassium acetate, 10% glycerol
- Add salts, allowing each to enter solution before adding the next.
- Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base.
- Filter through a 0.2 uM filter & store at 4oC.
Nicole/Andrew protocol for Chemically Competent cells
Materials and reagents
- E. coli line (Top 10, S17-1, BL21-AL, BL21-D3, JM109, Qiagen)
- TFB I (transformation buffer)
- TFB II
- TFB I (100 ml)
- 30 mM acetate K (0.294 g)
- 100 mM RbCl (1.21 g)
- 10 mM CaCl2 (0.14 g)
- 50 mM MnCl2 (1.0 g)
- 15% glycerol (15 ml)
- dH2O
- pH = 5.8 (use acetic acid to adjust)
- TFB II 100 ml
- 10 mM MOPS (0.21 g)
- 75 mM CaCl2 (1.1 g)
- 10 mM RbCl (0.12 g)
- 15% glycerol (15 ml)
- dH2O
- pH = 6.5 (use KOH to adjust)
Protocol
- 2 days before making cells, streak out the line of E. coli to make on LB plates (+strep for Top 10 and S17-1)
- 1 day before:
- inoculate 4 white capped test tubes or disposable 14 ml clear-top falcon tubes with 1 ml of LB (+strep)
- freeze appropriate color, autoclaved epi tubes in -80°C (80+ tubes)
- - white tube = Top 10
- - yellow tube = S17-1
- - pink tube = BL21-AL
- - purple tube = BL21-D3
- - green tube = JM109
- - blue tube = Qiagen
- Grow cells in 5 ml LB (+5 ul strep for Top 10 and S17-1 cells) overnight
- Transfer 1 ml of cells to 50 ml LB (use falcon tube) and grow at 37°C for 90 min
- - want OD of 0.4 or 0.5 before starting next steps
- Place on ice (0°C) for 1 min
- Spin at 6000g, 0°C for 5 min
- Add 15 ml cold dH2O
- Spin at 6000g, 0°C for 5 min, pour off super
- Add 10 ml cold TFB I to pellet
- Incubate on ice for 15 min
- Spin at 6000g, 0°C for 5 min, pour off super
- Add 1 ml cold TFB II to pellet
- Incubate on ice for 30 min
- Aliquot 50 ul into -80°C epi tubes (or into tubes sitting in dry ice)
- Immediately store at -80°C
- Best method
- take tubes out of freezer
- open all caps
- pipette 50 ul into each
- close caps
- back in -80°C
- VERY QUICKLY!
TEST CELLS BEFORE STOCKING FOR GENERAL USE
- For contamination
- Scrape a sample from frozen stock
- Streak on LB (no abx)
- Grow at 37°C overnight
- Check for contamination (E. coli should be translucent and yellowish) – If none is present test competency
- For competency
- Use PCM184 plasmid stock (and Amp or Kan/Tet)
- Follow protocol for transformation
Electrocompetent Cells
- You can make your own electro-competent cells for electroporation.
- Grow a small (~ 2 mL) overnight culture with appropriate antibiotic(s)
- Use overnight to inoculate: generally use ~ 200 uL/50 mL
- Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 2-3 times with 10% glycerol (everything on ice).
- Resuspend the pellet in a small volume, ideally up to 1/500ths of the culture volume.
- See graph below. Janet did this experiment. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable.
- Aliquot into 1.5 ml centrifuge tubes (0.1 ml in each), then flash freeze with liquid nitrogen. Store at -80C.
- Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See this page. Would allow you to calculate efficiency.