Team:Arizona State/Lab/Protocols/Transformation

NEB ([http://www.neb.com/nebecomm/products/protocol117.asp 10-beta] and [http://www.neb.com/nebecomm/products/protocol78.asp Turbo])

1. Thaw a tube of NEB Turbo/NEB 10-beta Competent E.coli cells until the last ice crystals disappear. Mix gently and carefully pipette 50 uL of cells into a transformation tube on ice.
2. Add 1-5 uL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42 C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 uL of room temperature SOC into the mixture.
7. Place at 37 C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plate to 37 C.
9. Spread 50-100 uL of each well-mixed dilution onto a selection plate and incubate 8-12 hours to overnight at 37 C. Alternatively, incubate at 30 C for 16 hours or 25 C for 24 hours.

[http://openwetware.org/wiki/Transforming_chemically_competent_cells TSS]

1. Thaw 50ul prepared competent TSS cells on ice.
2. Add approximately 1 ul prepped plasmid to the thawed cells. Make sure the culture is thoroughly mixed.
3. Incubate for 30 minutes on ice.
4. Incubate for 30 seconds at 42C (in a water bath).
5. Incubate for 2 minutes on ice.
6. Add 1 ml room temperature SOC.
7. Incubate for 1 hour at 37C with shaking.
8. Plate with appropriate antibiotic.
9. Grow overnight at 37C.