Er2796

A pCS2+ plasmid containing flag-tagged mgfp5 cloned into the EcoRI/XhoI sites (a gift from Lea Starita) was transformed into two strains of competent E. coli, ER2796 (from NEB, see “Preparation of calcium competent ER2796 E. coli”) and NEB 5-alpha (NEB cat. no. C2987I), using a standard heat shock method. 50 μL of the chemically competent cells were thawed on ice and mixed with 50 ng of plasmid DNA. The mixture was placed on ice for 30 minutes before being heat shocked at 42°C for 30 seconds. After heat shock, the cells were immediately placed back on ice for 5 minutes. Following incubation, 950 μL of room temperature SOC media (NEB cat. no. B9020S) was added, and the cells were allowed to outgrow for 1 hour in the absence of selection. 50 μL of the outgrown cells were diluted in 5 mL of selective media and grown overnight with shaking at 37°C and 220 rpm. Specifically, the NEB 5-alpha cells were grown in LB media with 100 μg/mL ampicillin, while the ER2796 cells were grown in LB media with 50 μg/mL kanamycin + 100 μg/mL ampicillin. The following day, plasmid DNA was extracted from the bacterial cells using the Monarch Plasmid Miniprep Kit (NEB cat. no. T1010L) following the manufacturer’s protocol. The elution of plasmid DNA was done with sterile water and the concentration was measured using the Qubit 1X dsDNA HS Assay Kit (Invitrogen cat. no. Q33231).