FAQ 12: How should I maintain the recombineering strains?

Answer 12: We recommend that you make a glycerol stock of all strains we send you ASAP. When you receive your strains, take a sterile loop and plunge it into the center of your stab. Good practice is to now streak it out for a single colony on a selective plate. For example if we sent you a strain containing pSIM5, steak out on a L+Cm (20µg/ml) agar plate or if the strain contains no drug resistance, an LB plate will do. Remember to incubate the plate at 32°!  Next grow a liquid culture (at 32°) from a single colony. Include the appropriate antibiotic (concentrations shown in FAQ 1) if the strain contains a plasmid. A glycerol can be made by adding 200µl of 50% glycerol to 700µl of an overnight culture of your strain in a small vial. These should be stored at -70°C. When you need the strain again, use a sterile loop to scrape out a small amount of frozen cells and streak it out for single colonies on an agar plate and incubate at 32°.