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From 2012.igem.org

I. Began growing DH5-α cells in a medium

1. Dilute one colony scraped from an agar plate in 5 mL of LB in a 25 or 50 mL flask and incubate overnight in a shaker at 37C (making competent DH5-α cells cont.)

II. Transformation of BL21 cells

1. Follow all steps from Preparation of Competent Origami on 6/5, except using a BL21 aliquot.

NOTES:

1. Many -80C aliquots of either strain will have a growth lag time of additional 2 or 3 hours. If unable to complete the procedure in one day, the growing cell solution may be allowed to saturate overnight, and then diluted to no more than OD = .1 After this, the solution’s OD should be checked regularly.
2. A good way to keep eppendorf tubes chilled during procedure is to freeze a closed container of them and their rack in -80C, only taking them out before procedure and keeping everything on ice.
3. Also, an efficient way to shock cells with liquid nitrogen is to pour liquid nitrogen in a plastic cup, empty the eppendorf tubes in a dish after drying their rack and further dry with a paper towel, dump the tubes in the liquid nitrogen, dump liquid nitrogen with the tubes back into dish, and quickly transfer the tubes into a labeled container.

III. Vacuumed, autoclaved, and kept records during waiting time.