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Team:Washington/Protocols/Transformation
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=General Transformation Protocol= | =General Transformation Protocol= | ||
| - | #Thaw 20 μl | + | #Thaw 20 μl of frozen competent cells on ice. Do not use glass tubes, which adsorb DNA. |
#Add 20 μl DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume) | #Add 20 μl DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume) | ||
#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 | + | #Incubate cells for 45-90 seconds at 42°C. |
#Incubate cells on ice for 2 min. | #Incubate cells on ice for 2 min. | ||
#Add 200mL of room temperature TB. | #Add 200mL of room temperature TB. | ||
#Incubate for 1 hour at 37°C on shaker. | #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 | + | ::Note: Step can be eliminated if plating on Amp plates, but not 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. | ||
Latest revision as of 02:42, 16 September 2011
General Transformation Protocol
- Thaw 20 μl of frozen competent cells on ice. Do not use glass tubes, which adsorb DNA.
- Add 20 μl 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 45-90 seconds at 42°C.
- Incubate cells on ice for 2 min.
- Add 200mL of room temperature TB.
- 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 other antibiotics
- Spread 100-300 μl onto a plate made with appropriate antibiotic.
- Grow overnight at 37°C.
Adapted from OpenWetWare protocol.
