Lidstrom:Competent Cell Preparation

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==== Supplies needed: ====
==== Supplies needed: ====
* Plate of E. Coli colonies
* Plate of E. Coli colonies
-
* SOB-Mg growth medium
+
* SOB-Mg growth medium
 +
** '''"-" is "minus", indicating that there is no added Mg'''
* Sterilized 500 mL Erlenmeyer flasks
* Sterilized 500 mL Erlenmeyer flasks
* 50 mL screw-cap polypropylene tubes
* 50 mL screw-cap polypropylene tubes
* Freezer tubes (1.5 mL)
* Freezer tubes (1.5 mL)
* SOC medium for recovery after heat shock (LB/TB is fine instead)
* SOC medium for recovery after heat shock (LB/TB is fine instead)
 +
==== Method ====
==== Method ====
# Pick several colonies off a freshly streaked plate into ~1 mL SOB-Mg growth medium
# 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.  
## 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.  
## Use more inoculum in the next step if cultures weren't grown overnight.  
-
# Inoculate 50 mL SOB-Mg growth medium with this culture.  Use between 1 uL to 1 mL stationary phase culture per mL of medium.  
+
# 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, 37<sup>o</sup>C until OD<sub>600</sub> is about 0.3, which corresponds to ~ 5*10<sup>7</sup>cells/mL
# Incubate at 275 rpm, 37<sup>o</sup>C until OD<sub>600</sub> is about 0.3, which corresponds to ~ 5*10<sup>7</sup>cells/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
# 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 4<sup>o</sup>C.  Decant the supernatant and invert tubes to remove excess culture medium.
# Pellet the cells at 750 - 1,000g (2500 rpm) for 14 min at 4<sup>o</sup>C.  Decant the supernatant and invert tubes to remove excess culture medium.
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# Flash freeze with liquid nitrogen  
# Flash freeze with liquid nitrogen  
# Store at -80oC to preserve them for many months
# Store at -80oC to preserve them for many months
 +
====Recipes:====
====Recipes:====
-
*SOB-Mg growth medium
+
 
-
*CCMB
+
== SOB-Mg growth medium (1 Liter) ==
 +
 
 +
{|border="2" cellpadding="1"
 +
|+Setting cell widths
 +
!width="120"|Ingredient
 +
!width="60"|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 (1 Liter) ==
 +
 
 +
{|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===
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# Grow cells in 5 ml LB (+5 ul strep for Top 10 and S17-1 cells) overnight
# 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  
+
# Transfer 1 ml of cells to each 50 ml LB flask and grow at 37°C for 90 min  
#:- want OD of '''0.4 or 0.5''' before starting next steps
#:- want OD of '''0.4 or 0.5''' before starting next steps
# Place on ice (0°C) for 1 min
# Place on ice (0°C) for 1 min
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==Electrocompetent Cells==
==Electrocompetent Cells==
-
*You can make your own electro-competent cells for electroporation.
+
*You can easily make your own electro-competent cells for electroporation.
#Grow a small (~ 2 mL) overnight culture with appropriate antibiotic(s)  
#Grow a small (~ 2 mL) overnight culture with appropriate antibiotic(s)  
#Use overnight to inoculate: generally use ~ 200 uL/50 mL  
#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).
+
#Let grow for about 3-4 h (OD 0.4-0.6 or so)
-
#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.  
+
## If using the tube spec, the path length is longer than in the cuvettes. Divide tube specs by 1.65 to convert to the 1 cm path length OD.  (If using tube spec, let the OD get to ~1.)
-
## See graph below.  [[User:Janet B. Matsen|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.  
+
# Centrifuge and wash 2-3 times with 10% glycerol (everything on ice).
-
#Aliquot into 1.5 ml centrifuge tubes (0.1 ml in each), then flash freeze with liquid nitrogen.  Store at -80C.
+
## Once or twice is fine.  Don't waste time/energy doing much more.  -[[User:Janet B. Matsen|JM]] 8/2013
-
*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care.  See [[Electrocompetent_Cells| this page]].  Would allow you to calculate efficiency.
+
# ''' Remove pretty much all of the supernatant after the last centrifugation '''
-
[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg|thumb|center|electroporation: number of colonies versus competent cell density]]
+
## Leave just enough water to allow for pipetting.  The volume of supernatant left should be < 1/500<sup>ths</sup> of the culture volume.  
 +
## See [[User:Janet B. Matsen|Janet's]] graph below.  The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable.  [[image:2012_11 electroporation - number of colonies versus competent cell density.jpg|thumb|center|upright=2.0|''E. coli'' electroporation: number of colonies versus competent cell density.  Note the log scale.]]
 +
###Each electroporation used 0.2 ng of pGA3K3 RFP DNA.  ([https://docs.google.com/document/d/1btKWgYd1GemMa2mBmg-U72bnu2knSyjf_yGqXo8P1nY/edit experiment details], [https://docs.google.com/spreadsheet/ccc?key=0AlVxrZi130nMdDk0VHUxWHB5bzdzUVhCNnczVlkyLWc#gid=2 experiment planning spreadsheet])
 +
### Undiluted cells in this experiment are actually not very concentrated.  Glycerol water of volume 1/500th of the culture volume was mixed back in.  [[User:Janet B. Matsen|Janet]] has subsequently (8/2013) learned that more concentrated is much better, and is now routinely getting lawns from gibson assembly transformations.  
 +
#Aliquot into 1.5 ml centrifuge tubes (40-50uL 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 [[Electrocompetent_Cells| this page]].  
 +
* [[User:Janet B. Matsen|Janet's] cells clump into a "booger" during recovery (pre-plating) that was mostly unspreadable.  The clumping is less of an issue if you don't centrifuge them before plating. For this reason I recover in 200 uL and plate all of it.

Revision as of 14:39, 6 September 2013

Back to Protocols


Contents

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
    • "-" is "minus", indicating that there is no added Mg
  • 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

  1. Pick several colonies off a freshly streaked plate into ~1 mL SOB-Mg growth medium
    1. Grow cells overnight or several hours in media with appropriate antibiotics if available.
    2. Use more inoculum in the next step if cultures weren't grown overnight.
  2. Inoculate 50 mL SOB-Mg growth medium with this culture. Use 500 uL of stationary phase culture for 50 mL SOB-Mg medium.
  3. Incubate at 275 rpm, 37oC until OD600 is about 0.3, which corresponds to ~ 5*107cells/mL
    1. Higher OD isn't usually a problem for routine work.
  4. Collect in sterile 50 mL polypropylene centrifuge tube(s) and chill on ice for 10 minutes
  5. 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.
  6. Disperse cells in ~1/3 volume of CCMB by gentle vortexing or rapping of the centrifuge tube.
  7. Incubate on ice for 20 minutes
  8. Centrifuge at 2500 rpm for 10 min at 4oC
  9. Resuspend cells in CCMB at 1/12 the original culture volume
  10. Make aliquots in eppendorf tubes, ideally on ice
  11. Flash freeze with liquid nitrogen
  12. Store at -80oC to preserve them for many months

Recipes:

SOB-Mg growth medium (1 Liter)

Setting cell widths
Ingredient Amount
Bacto Tryptone20g
Bacto Yeast Extract5g
1M NaCl10mL
1M KCl2.5mL
  • Add water to make 1L
  • Autoclave
  • Dispense into smaller bottles for lower contamination risk

CCMB (1 Liter)

Setting cell widths
Ingredient Amount Final Concentration
Potassium Acetate, 1M, pH 710 mL10mM
Glycerol100g10% (w/v)
CaCl2.2H2O11.8g80mM
MnCl2.4H2O4g20mM
MgCl2.6H2O2.5mL10mM
  1. Prepare a 1M solution of potassium acetate, pH 7.0 using KOH.
    1. Filter through a 0.2 uM membrane & store frozen
  2. Prepare a solution of 10% potassium acetate, 10% glycerol
  3. Add salts, allowing each to enter solution before adding the next.
  4. Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base.
  5. 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


  1. Grow cells in 5 ml LB (+5 ul strep for Top 10 and S17-1 cells) overnight
  2. Transfer 1 ml of cells to each 50 ml LB flask and grow at 37°C for 90 min
    - want OD of 0.4 or 0.5 before starting next steps
  3. Place on ice (0°C) for 1 min
  4. Spin at 6000g, 0°C for 5 min
  5. Add 15 ml cold dH2O
  6. Spin at 6000g, 0°C for 5 min, pour off super
  7. Add 10 ml cold TFB I to pellet
  8. Incubate on ice for 15 min
  9. Spin at 6000g, 0°C for 5 min, pour off super
  10. Add 1 ml cold TFB II to pellet
  11. Incubate on ice for 30 min
  12. Aliquot 50 ul into -80°C epi tubes (or into tubes sitting in dry ice)
  13. 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
  1. Scrape a sample from frozen stock
  2. Streak on LB (no abx)
  3. Grow at 37°C overnight
  4. 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 easily make your own electro-competent cells for electroporation.
  1. Grow a small (~ 2 mL) overnight culture with appropriate antibiotic(s)
  2. Use overnight to inoculate: generally use ~ 200 uL/50 mL
  3. Let grow for about 3-4 h (OD 0.4-0.6 or so)
    1. If using the tube spec, the path length is longer than in the cuvettes. Divide tube specs by 1.65 to convert to the 1 cm path length OD. (If using tube spec, let the OD get to ~1.)
  4. Centrifuge and wash 2-3 times with 10% glycerol (everything on ice).
    1. Once or twice is fine. Don't waste time/energy doing much more. -JM 8/2013
  5. Remove pretty much all of the supernatant after the last centrifugation
    1. Leave just enough water to allow for pipetting. The volume of supernatant left should be < 1/500ths of the culture volume.
    2. See Janet's graph below. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable.
      E. coli electroporation: number of colonies versus competent cell density.  Note the log scale.
      E. coli electroporation: number of colonies versus competent cell density. Note the log scale.
      1. Each electroporation used 0.2 ng of pGA3K3 RFP DNA. (experiment details, experiment planning spreadsheet)
      2. Undiluted cells in this experiment are actually not very concentrated. Glycerol water of volume 1/500th of the culture volume was mixed back in. Janet has subsequently (8/2013) learned that more concentrated is much better, and is now routinely getting lawns from gibson assembly transformations.
  6. Aliquot into 1.5 ml centrifuge tubes (40-50uL 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.
  • [[User:Janet B. Matsen|Janet's] cells clump into a "booger" during recovery (pre-plating) that was mostly unspreadable. The clumping is less of an issue if you don't centrifuge them before plating. For this reason I recover in 200 uL and plate all of it.
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