Protocol

1. ON ICE: If frozen, thaw the 200 μL TSS prepared competent cells on ice.

2. 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.

3. ON ICE: Add 1 μL of plasmid DNA to each 100 μL transformation reaction.

4. ON ICE: Allow transformations to chill for 60 min.

5. Add 900 μL of room temperature liquid medium to each transformation.

6. Incubate transformations at 37°C with rotation for 60 min.

-Remember to pre-warm the solid medium during this time.

7. Using pre-warmed solid selective medium (LB or SOC), plate out 100 μL and incubate at 37°C overnight (~16-18 h).

8. Verify successful transformations as necessary.