Lidstrom:Competent Cell Preparation

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(Nicole/Andrew protocol for Chemically Competent cells)
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*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.
*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.
-
==Electrocompetent Cells==
+
==Amanda/Janet Protocol for Chemically Competent Cells ==
-
*You can make your own electro-competent cells for electroporation.
+
=== Supplies needed: ===
-
**From Amada's past mentor in undergrad:
+
* Plate of E. Coli colonies
-
***Grow the cells overnight
+
* SOB-Mg growth medium
-
***Inoculate from this the next morning: generally use 200 uL/50 mL  
+
* Sterilized 500 mL Erlenmeyer flasks
-
***Let grow for about 3-4 h (OD 0.4-0.6 or so),  then centrifuge and wash 3 times with 10% glycerol (everything on ice).
+
* 50 mL screw-cap polypropylene tubes
-
***Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
+
* Freezer tubes (1.5 mL)
-
****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.
+
* SOC medium (LB/TB is fine instead)
 +
=== Method ===
 +
# Pick several colonies off a freshly streaked plate into 1 mL SOB-Mg growth medium
 +
## Grow overnight in media with appropriate antibiotics if available
 +
# Inoculate 50 mL SOB-Mg growth medium with this cultureUse between 1 uL to 1 mL stationary phase culture per mL of medium.  
 +
#
 +
 
Line 87: Line 93:
:Use PCM184 plasmid stock (and Amp or Kan/Tet)
:Use PCM184 plasmid stock (and Amp or Kan/Tet)
:Follow protocol for transformation
:Follow protocol for transformation
 +
 +
==Electrocompetent Cells==
 +
*You can make your own electro-competent cells for electroporation.
 +
**From Amada's past mentor in undergrad:
 +
***Grow the cells overnight
 +
***Inoculate from this the next morning: generally use 200 uL/50 mL
 +
***Let grow for about 3-4 h (OD 0.4-0.6 or so),  then centrifuge and wash 3 times with 10% glycerol (everything on ice).
 +
***Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and 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]].  Would allow you to calculate efficiency.

Revision as of 19:47, 19 October 2012

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
  • Sterilized 500 mL Erlenmeyer flasks
  • 50 mL screw-cap polypropylene tubes
  • Freezer tubes (1.5 mL)
  • SOC medium (LB/TB is fine instead)

Method

  1. Pick several colonies off a freshly streaked plate into 1 mL SOB-Mg growth medium
    1. Grow overnight in media with appropriate antibiotics if available
  2. Inoculate 50 mL SOB-Mg growth medium with this culture. Use between 1 uL to 1 mL stationary phase culture per mL of medium.


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 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
  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 make your own electro-competent cells for electroporation.
    • From Amada's past mentor in undergrad:
      • Grow the cells overnight
      • Inoculate from this the next morning: generally use 200 uL/50 mL
      • Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice).
      • Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and 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.
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