TSS Chemical Transformation
Updated 7/8/2019 02:37 AM
Introduction
Materials
- Equipment
- Incubator
- Selection plates
- Microcentrifuge tubes
- Ice (w/styrofoam box)
- Reagents
- TSS competent cells (in Eppendorf tubes)
- SOC media
- Plasmid DNA
Procedure
Important Information to be documented
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Important Notes
- The first part of this procedure should be done on ice. In the cooler box, it might freeze, but you can put tubes on top of the box tube slots for the same effect.
- It is recommended to thaw out the plasmid on ice as well.
- The recommended amount of DNA is 10pg-100ng for 50µL of competent cells. With samples at ~1ng/µL, 1µL is ok.
Procedure
- Thaw tubes of competent cells in 4C fridge for 10 minutes.
- OPTIONAL: Pipette 100µL each into a new microcentrifuge tube.
CRITICALPipetting live cells should be done SLOWLY and carefully as to not harm them.
- Add 1ng of plasmid DNA to corresponding tubes. Carefully flick the tubes 4–5 times to mix cells and DNA. Centrifuge down with a tabletop centrifuge as necessary.
- Place the mixture on ice for 1 hour. Do not mix.
- Pipette 900µL of room temperature SOC into each mixture.
This should be done in the flow hood under sterile conditions.
- Incubate at 37°C for 1 hour at 250 rpm.
Shaky is already set to this temperature and rpm.
- Pre-warm selection plates to 37°C while tubes are being incubated.
- Plate 200µL of transformation onto a proper selection plate.
- Incubate for 24 hours at 37°C.
Transformation into TSS-prepared competent cells.