Team:Exeter/lab book/proto/1

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Protocol 1


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase

Transformation Protocol from Invitrogen, for Top10 Competent Cells
Materials Supplied by the User You will need the following items for transformation: 37°C shaking and non-shaking incubator 10 cm diameter LB agar plates with appropriate antibiotic Ice bucket with ice 42°C water bath Before Starting Equilibrate a water bath to 42°C Warm the vial of S.O.C medium to room temperature Spread X-Gal onto LB agar plates with antibiotic, if desired for blue/white selection Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation) Important: It is essential that LB plates containing 100 mg/ml ampicillin are pre-warmed if you are performing the rapid chemical transformation procedure. Chemical Transformation Procedure The instructions provided below are for general use. Specific instructions for particular applications such as Zero Blunt® PCR Cloning are provided in the manual for that kit. 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 to 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 µl to 200 µl from each transformation vial on separate, labelled 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.

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