Transforming chemically competent cells (Inoue): Difference between revisions
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#Incubate on ice for 30 minutes | #Incubate on ice for 30 minutes | ||
#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min. | #*Note: If you are in a rush, you can shorten this incubation time to 5-10 min. | ||
#Incubate cells for 30 seconds at | #Incubate cells for 30 seconds at 42°C. | ||
#Incubate cells on ice for 2 min. | #Incubate cells on ice for 2 min. | ||
#Add 4 volumes of room temperature [[SOC]] (not critical) | #Add 4 volumes of room temperature [[SOC]] (not critical) | ||
#Incubate for 1 hour at | #Incubate for 1 hour at 37°C on shaker. | ||
#*Note: Can also save some time here by reducing incubation to ~45 min. | #*Note: Can also save some time here by reducing incubation to ~45 min. | ||
#*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics | #*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics | ||
#Spread 100-300 μl onto a plate made with appropriate antibiotic. | #Spread 100-300 μl onto a plate made with appropriate antibiotic. | ||
#Grow overnight at 37 °C. | #Grow overnight at 37°C. | ||
[[Category:E.Coli Protocol]] | [[Category:E.Coli Protocol]] | ||
Revision as of 05:28, 12 April 2006
Also see Preparing chemically competent cells (Inoue)
Method
- Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
- Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
- Incubate on ice for 30 minutes
- Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
- Incubate cells for 30 seconds at 42°C.
- Incubate cells on ice for 2 min.
- Add 4 volumes of room temperature SOC (not critical)
- Incubate for 1 hour at 37°C on shaker.
- Note: Can also save some time here by reducing incubation to ~45 min.
- Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
- Spread 100-300 μl onto a plate made with appropriate antibiotic.
- Grow overnight at 37°C.
