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 .
