Team:Michigan/Notebook/TransformationProtocol

Transformation Standard Protocol

Transformation of Plasmids (using heat shock)

1. Precool 1.5 mL tubes, 1 for each transformation and 1 for a positive control
2. Defrost competent cells (usually XL1 blue) on ice
3. Mix 100 μL (or 50 μL if doing many transformations) of cells with 1-2 μL of prepped plasmid (or 10 μL of plasmid if coming from a ligation)
4. Incubate mixture on ice for 30 minutes
5. Heat shock at 42 degrees Celsius for 45 seconds
6. Incubate on ice for 5 min
7. Add 900 μL pre-warmed LB media & allow to recover on spinning wheel in incubator (37 degrees Celsius) for 1 hour
8. Spin down and resuspend in 50 μL LB media
9. Selectively plate and grow in incubator overnight

Transformation of Linearized DNA via Electroporation

This technique was used for crossing our linearized landing pad plasmids onto the chromosome.

1. Add 1-2 μL of DNA to 50 μL of electrocompetent cells into electroporation tube
2. Electroporate (using BIORAD Micropulser) and immediately add 0.9 mL of LB media
3. Incubate cells for 1 hour to recover
4. Spin down and resuspend in 100 μL of LB
5. Selectively plate and grow in incubator overnight