One Shot® TOP10 Chemically Competent E. coli Transformation
Centrifuge the vial(s) containing the ligation reaction(s) briefly and place on ice.
Thaw, on ice, one 50 μL vial of One Shot® cells for each ligation/transformation.
Pipet 1–5 μL of each ligation reaction directly into the vial of competent cells and mix by tapping gently.
Do not mix by pipetting up and down.
The remaining ligation mixture(s) can be stored at −20°C.
Incubate the vial(s) on ice for 30 minutes.
Incubate for exactly 30 seconds in the 42°C water bath.
Do not mix or shake.
Remove vial(s) from the 42°C bath and place them on ice.
Add 250 μL of pre-warmed S.O.C medium to each vial.
S.O.C is a rich medium; sterile technique must be practiced to avoid contamination.
Place the vial(s) in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s).
Shake the vial(s) at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.
Spread 20–200 μL from each transformation vial on separate, labeled LB agar plates.
The remaining transformation mix may be stored at 4°C and plated out the next day, if desired.
Invert the plate(s) and incubate at 37°C overnight.
Select colonies and analyze by plasmid isolation, PCR, or sequencing.
