Team:UNICAMP-EMSE Brazil/protocols/Transforming bacteria chemo

Transforming bacteria: Chemo-transformation

To transform the CaCl2-treated cells directly, transfer 200 μl of each suspension of competent cells to a sterile, chilled 17 x 100-mm polypropylene tube using a chilled micropipette tip. Add DNA (no more than 50 ng in a volume of 10 μl or less) to each tube. Mix the contents of the tubes by swirling gently. Store the tubes on ice for 30 minutes.
Transfer the tubes to a rack placed in a preheated 42°C circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes.
Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 1-2 minutes.
Add 800 μl of SOC medium (can use LB) to each tube. Incubate the cultures for 45 minutes in a water bath set at 37°C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.

Transfer the appropriate volume (up to 200 μl per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic (can use LB).
Store the plates at room temperature until the liquid has been absorbed. Invert the plates and incubate at 37°C. Transformed colonies should appear in 12-16 hours.

Using sterile handling pick a colony with the wood stick and inoculate it in a falcon tube containing 4 mL of LB medium and the appropriate antibiotics.
Label the tube with bacteria, media, antibiotic, plasmid and gene, number of the plate, number of the colony, date and your name.
Incubate for 16 h at 150-200 rpm and 37°C.