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Team:Northwestern/Notebook/Protocols/Transformation
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| - | #Thaw cells on ice | + | |
| - | + | ||
| - | #* | + | #Thaw chemically competent cells on ice. Be very careful - do not thaw with hands (in fact, bring the ice bucket to the freezer when you're getting them out) because the cells will have dramatically lower transformation efficiency. While thawing, prepare DNA from well plates: |
| - | # | + | #*With a pipette tip, punch a hole through the foil cover into the corresponding well to the part that you want. Make sure you have properly oriented the plate (label rows/columns with a sharpie if it's the first time using the plate). Transfer excess DNA from well to a microcentrifuge tube and store at -20 °C. |
| - | # | + | #*Add 10uL of nuclease free H2O. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. |
| - | #Incubate cells for | + | #Pipette 25uL aliquots of cells into overnight tubes on ice. Add 1 uL DNA, then pipette gently to mix. Let sit for at least 30 minutes on ice. |
| + | #Incubate cells for 40 seconds at 42 °C (use a stopwatch for exact timing). | ||
#Incubate cells on ice for 2 min. | #Incubate cells on ice for 2 min. | ||
| - | #Add 200 uL LB | + | #Add 200 uL LB. |
| - | #Incubate for 1 hour at | + | #Incubate for 1 hour at 37 °C in shaker. If you need to cut corners, for Amp you can incubate for 5-10 minutes if you are in a hurry. For other antibodies, you can probably get away with about 40 minutes). |
| - | #Spread | + | #Spread 200uL onto a plate made with the appropriate antibiotic. |
| - | #Grow overnight at | + | #Grow overnight at 37 °C. |
| - | + | ||
| - | Adapted from [http://openwetware.org/wiki/Transforming_chemically_competent_cells OpenWetWare] | + | Adapted from [http://openwetware.org/wiki/Transforming_chemically_competent_cells OpenWetWare] and [http://partsregistry.org/Help:Spring_2011_DNA_distribution The Parts Registry] |
Latest revision as of 15:30, 23 August 2011
PROJECT
RESULTS
CONSIDERATIONS
ABOUT US
NOTEBOOK
ATTRIBUTIONS
Transformation
- Thaw chemically competent cells on ice. Be very careful - do not thaw with hands (in fact, bring the ice bucket to the freezer when you're getting them out) because the cells will have dramatically lower transformation efficiency. While thawing, prepare DNA from well plates:
- With a pipette tip, punch a hole through the foil cover into the corresponding well to the part that you want. Make sure you have properly oriented the plate (label rows/columns with a sharpie if it's the first time using the plate). Transfer excess DNA from well to a microcentrifuge tube and store at -20 °C.
- Add 10uL of nuclease free H2O. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.
- Pipette 25uL aliquots of cells into overnight tubes on ice. Add 1 uL DNA, then pipette gently to mix. Let sit for at least 30 minutes on ice.
- Incubate cells for 40 seconds at 42 °C (use a stopwatch for exact timing).
- Incubate cells on ice for 2 min.
- Add 200 uL LB.
- Incubate for 1 hour at 37 °C in shaker. If you need to cut corners, for Amp you can incubate for 5-10 minutes if you are in a hurry. For other antibodies, you can probably get away with about 40 minutes).
- Spread 200uL onto a plate made with the appropriate antibiotic.
- Grow overnight at 37 °C.
Adapted from [http://openwetware.org/wiki/Transforming_chemically_competent_cells OpenWetWare] and [http://partsregistry.org/Help:Spring_2011_DNA_distribution The Parts Registry]
