Team:Valencia/LB Agar

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Transforming Chemically Competent Cells E.Coli DH5α-T1^R

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

1. Briefly centrifuge the ligation reaction and place on ice.
2. Thaw, on ice, one 50 µl vial of One Shot cells for each ligation/transformation.
3. 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.
4. Incubate the vial on ice for 30 minutes.
5. Incubate for exactly 30 seconds in the 42ºC water bath. Do not mix or shake.
6. Remove vial from the 42ºC bath and place on ice.
7. Add 250 µl of pre-warmed SOC medium to each vial. (SOC is a rich medium; sterile technique must be practiced to avoid contamination.)
8. 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.
9. Spread 20 µl to 200 µl from each transformation vial on separate, labeled LB agar plates. We recommend that you plate two different volumes.
10. Store the remaining transformation reaction at +4ºC and plate out the next day, if desired.
11. Invert the plates and incubate at 37ºC overnight.
12. Select colonies and analyze by plasmid isolation, PCR, or sequencing.