TSS Competent E. coli Transformation
Updated 6/12/2015 08:41 PM
Introduction
Materials
- Consumables
- Ice
- TSS Competent E. coli
- Liquid non-selective medium (LB or SOC)
- Solid selective medium (LB or SOC)
- Equipment
- Incubator (must control temperature at 37°C)
- Disposables
- 1.5mL PP tube (snap-cap)
Procedure
Protocol
- ON ICE: If frozen, thaw the 200 μL TSS prepared competent cells on ice.
- ON ICE: Transfer 100 μL to a pre-chilled 1.5mL PP tube (snap-cap). There will be 100 μL of TSS competent cells per transformation.
CRITICALEnsure cells are well mixed! Mix via gentle thumping/pipette aspiration.
-It is suggested that one of the two aliquots be used for a negative control.
- ON ICE: Add 1 μL of plasmid DNA to each 100 μL transformation reaction.
- ON ICE: Allow transformations to chill for 60 min.
- Add 900 μL of room temperature liquid medium to each transformation.
- Incubate transformations at 37°C with rotation for 60 min.
-Remember to pre-warm the solid medium during this time.
- Using pre-warmed solid selective medium (LB or SOC), plate out 100 μL and incubate at 37°C overnight (~16-18 h).
- Verify successful transformations as necessary.
This method of transformation is to be used on competent cells prepared following the TSS Competent E. coli Preparation protocol.
NOTE: This method has been successful for transforming pUC18 into BLR(DE3) with an efficiency >3E5 CFU μg-1. Additionally, a vector >8kb was transformed into BLR(DE3) prepared using the protocol above with efficiencies of 1E3 CFU μg-1 to 1E4 CFU μg-1.