Lidstrom:Competent Cell Preparation: Difference between revisions
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*You need fresh cells. | *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. | **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. | **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. | *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, 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 | |||
# 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. | |||
# 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 4<sup>o</sup>C | |||
# 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 | |||
*CCMB | |||
===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 1/500<sup>ths</sup> of the culture volume. | |||
## 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. | |||
#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 [[Electrocompetent_Cells| this page]]. Would allow you to calculate efficiency. | |||
[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg|thumb|center|electroporation: number of colonies versus competent cell density]] |
Revision as of 18:12, 4 January 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
- CCMB
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.