Specify various methods for preparation of Competent cells and which bacterial strain will be good for this?
Dear Saurov,
For E.coli, the common prokaryotic expression host, chemical methods are available to prepare competent cells. These days, competent cells are commercially available, and they take up the DNA much efficiently. In Pichia pastoris (one of the eukaryotic systems), you may have to prepare them (competent cells) Lithium chloride or Zymolyase. If you are a little specific about your expression host and purpose, better suggestions could be given.
A comparison like this was done before. Here is a summary (chemical transformation hanahan's vs DMSO vs CaCl2):
Hanahan's for DH5α, XL-1 Blue and JM109 strains. CaCl2 method was best for SCS110, TOP10 and BL21 strains. The methods are in the ref.
ref:
Article A comparison and optimization of methods and factors affecti...
You will get better efficiencies using electroporation:
competent cells:
http://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells
electroporation:
https://openwetware.org/wiki/Richard_Lab:Electroporation_of_E._coli
SOP: Preparation of chemically competent E. coli cells
Reference:
SOP generated by: Mathias Klein 03.2014
Approved by:
Materials and Solutions
- E. coli strain (most often DH5α)
- LB agar plates
- LB medium
- sterile 10 ml glass tubes
- sterile 500 ml Erlenmeyer flasks
- ice-cold CaCl2 solution (100 mM CaCl2, 15% glycerol)
- bucket with liquid nitrogen
Method
- Streak E. coli cells for single colonies on an LB agar plate and incubate at 37°C overnight.
- Pick a single colony and inoculate 5 ml of LB medium in a 10 ml glass tube. Incubate at 37°C and 250 rpm overnight.
- Use 1 mL from the overnight culture to inoculate 50 ml of LB medium in a 500 ml Erlenmeyer flask. Grow the cells shaking at 37°C and 250 rpm until they reach an OD600 of 0.4-0.5 (OD must not be higher than 0.5!!).
- Fill the culture into a 50 mL falcon tube (clean bench!!) and place the tubes on ice for 20 min.
FROM THIS STEP ON IT IS CRUCIAL TO PERFORM ALL STEPS ON ICE! IF TUBES ARE REMOVED FROM THE ICE BUCKET EXPOSURE TO ROOM TEMPERATURE MUST BE AS SHORT AS POSSIBLE!
- Centrifuge at 3,000 g at 4°C (pre-cool the centrifuge!) for 15 min.
- Carefully remove the supernatant and resuspend the cell pellet in 15 ml of ice-cold CaCl2 solution (do not vortex, carefully shake and/or pipette up and down).
- Centrifuge at 2,500 g and 4°C for 10 min.
- Carefully remove the supernatant and resuspend the cell pellet in 10 ml of ice-cold CaCl2 solution (do not vortex, carefully shake and/or pipette up and down).
- Keep the cell suspension on ice for 30 min.
- Centrifuge again at 2,500 g and 4°C for 10 min.
- Remove the supernatant; carefully resuspend the cells in 1.2 mL of ice-cold CaCl2 solution. Aliquot 200 µL in pre-chilled 1.5 mL tubes. Immediately freeze cells in liquid nitrogen (be careful!) and freeze at -80°C.
Hello
I agree with Miguel Fernández-Niño, it's a perfect answer for you
Good Luck
- Badges
- Science topic