Thomas Wang
7.021 Section B
Out of class ex. #1
9/30/02
Methods for Experiment V-A “P1 Transduction – Stabilization and Mapping”
Bacterial strains used in this experiment are listed below. BW140 and C600 recipient strains were grown overnight in LB liquid media + 5mM CaCl2. One ml of each recipient was transferred into 6 Eppendorf tubes for a total of 12 tubes. Samples were centrifuged at 6000 rpm for 2 minutes, decanted, and then resuspended in .3 ml MC medium (10 mM MgSO4, 5mM CaCl2). For each of the P1 lysates (BK1, B1W1a, B1W1b, D3W1, K3W1), 10 ul were added to one BW140 recipient tube and one C600 recipient tube. The remaining C600 and BW140 sample were used as a control. All tubes were incubated on the bench for 30 minutes.
Each sample was transferred to sterile culture tubes containing 1 ml LB medium. 100 ul of 1M sodium citrate were added to the samples, and all 12 tubes were mixed on the roller drum at
370 C for 1 hour.
Cultures were transferred to fresh Eppendorf tubes, centrifuged at 6000 rpm for 2 min, decanted, and resuspended in 50 ul of LB + .1 M sodium citrate. Each of the 12 samples was then plated directly onto an LB Kan plate and incubated at 370 C. As an additional control, 5 ul spots of each of the 5 P1 lysates alone were plated on an LB Kan plate. After drying, the plate was incubated at 370 C as well.
Methods for Experiment II-A “Transposon Mutagenesis of an Ara+ strain of E. coli”
Bacterial strains used in this experiment are listed below. Lambda 1205 phage stock and pnK/BW140 culture were grown overnight in LBMM (LB + 10 mM MgSO4 + 0.2% Maltose) + tetracycline, diluted with LBMI (LB + 10 mM MgSO4 + 0.8 mM IPTG), grown to stationary phase, concentrated by centrifugation, and resuspended in 1/10 volume of LBMI (LB + 10 mM MgSO4 + 0.8 mM IPTG). As controls, 0.1 ml of cells and 0.1 ml of phage suspension were transferred into separate culture tubes. In a third tube, .5 ml of cell and .5 ml of phage stock were mixed gently. All three tubes were incubated without shaking at 370 C for 30 minutes.
10 ul of 1 M sodium citrate and 120 uL LB were added to each of the control tubes and 100 ul of 1 M sodium citrate and 1.2 ml LB were added to the third experimental tube. All three tubes were mixed on roller drums at 370 C for 1 hour.
105 ul of each of the controls were plated onto separate MacConkey Ara Kan plates. 105 ul of the infected pnK/BW140 culture were plated onto 1 LB X-gal plate and onto 18 Mac Ara Kan plates. Plates were allowed to dry and then incubated overnight at 370 C.