Team:Valencia/Transforming ecoli
From 2012.igem.org
Transforming Chemically Competent Cells E.Coli DH5α-T1R
Reagents and Materials
- 37ºC shaking and non-shaking incubator.
- 10 cm diameter LB agar plates with appropriate antibiotic.
- Ice bucket with ice.
- 42ºC water bath
Protocol
- Briefly centrifuge the ligation reaction and place on ice.
- Thaw, on ice, one 50 µl vial of One Shot cells for each ligation/transformation.
- Pipet 1 to 5 µl of each ligation reaction directly into the competent cells and mix by tapping gently. Do not mix by pipetting up and down. Store the remaining ligation reaction at -20ºC.
- Incubate the vial on ice for 30 minutes.
- Incubate for exactly 30 seconds in the 42ºC water bath. Do not mix or shake.
- Remove vial from the 42ºC bath and place on ice.
- Add 250 µl of pre-warmed SOC medium to each vial. (SOC is a rich medium; sterile technique must be practiced to avoid contamination.)
- Place the vial in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial. Shake the vial at 37ºC for exactly 1 hour at 225 rpm in a shaking incubator.
- Spread 20 µl to 200 µl from each transformation vial on separate, labeled LB agar plates. We recommend that you plate two different volumes.
- Store the remaining transformation reaction at +4ºC and plate out the next day, if desired.
- Invert the plates and incubate at 37ºC overnight.
- Select colonies and analyze by plasmid isolation, PCR, or sequencing.