Lidstrom:Competent Cell Preparation: Difference between revisions
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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. | ||
== | ==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 === | |||
# 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 culture. Use 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 17:47, 19 October 2012
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 (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 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
- 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.
- 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.
- From Amada's past mentor in undergrad: