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===I. Growth and Induction=== *1. Transform C3013 E. coli cells (NEB) (C3013I/H, plasmid, SOC, AMP/LB broth/agar/culture plates) (use +/- controls of puc1 and no transformation) **a. For C3013H: Thaw a tube of T7 Express lysY/Iq Competent E. coli cells on ice for 10 minutes. **b. For C3013I: Thaw a tube of T7 Express lysY/Iq Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. **c. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. **d. Place the mixture on ice for 30 minutes. Do not mix. **e. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix. **f. Place on ice for 5 minutes. Do not mix. **g. Pipette 950 µl of room temperature SOC into the mixture. **h. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. **i. Warm selection plates to 37°C. **j. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. '''(use half plate for each, 200 uL, 50 uL, 50 uL of 1:10, 50 uL of 1:100)'''' **k. Spread 50-100 µl of each dilution onto a selection plate (100 ug/ml amp) and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or at 25°C for 48 hours. *2. 1L cultures inoculated with ( colony grown up in 5ml O/N, inoculate 1L in morning) (1L LB culture broth, amp at 100 μg/ml, 100 mg per 1L) *3. 37C for 3hr to A600 ~ 0.9 (nanodrop to measure A600) *4. Add 1M IPTG to 0.25mM (IPTG) (250 uL of 1 M IPTG per 1 L culture?, 4000X dilution) *5. Chill cultures in cold water to about 10C *6. Grow 23C for 4hr to A600 ~ 3.0 *7. Centrifuge and freeze pellet -70C overnight and thaw in 80ml HEGX (BIG OL’ CENTRIFUGE,Scupula, Culture tubes 6000xg for 15 minutes) *STOPPING POINT
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