Cloning Cytochrome b PCR Fragment into pGEM-T - Cornell College

3. Incubate the plated cells overnight at 37˚C. Approximately 20-50 colonies per plate areroutinely seen. White colonies generally contain cloned insert DNA while blue colonies donot. Blue colonies should also be checked for cloned insert DNA.III.Plasmid Clone Isolation for Characterization using Zyppy Plasmid Miniprep KitA. E. coli Inoculation1. In order to determine if your transformants contain plasmids with PCR amplifiedmitochondria cytochrome b gene, inoculate a single white colony (blue colonies do notcontain PCR products) from your transformation plate into sterile liquid LB (10 mL)containing 50 g/mL of ampicillin. Inoculate up to 6 separate white colonies per plate(inoculated into 6 different tubes of LB; one colony per tube of LB). Grow thetransformants overnight at 37°C in the shaking incubator (200 RPM).LB liquid cultureBacto-tryptone10.0 g/LBacto-yeast5.0 g/LNaCl10.0 g/LAdd dH 2 O to 800 mL.Adjust the pH to 7.5Bring the volume to 1 LAliquot the liter of solution into appropriate volumesAutoclaveB. Buffer Preparations.Before beginning the protocol, make sure the following preparations have been completed.1. Add RNase A to the Neutralization Buffer: aliquot 1 mL of Neutralization Buffer into the tubecontaining the lyophilized RNase A, mix and transfer the solution back into the NeutralizationBuffer bottle. This buffer must be stored at 4° C.2. Add 24 mL of 100% ethanol (or 26 mL of 95% ethanol) to the 6 mL Zyppy Wash Bufferbefore use.3. The 7X Lysis Buffer may have a precipitate. If you notice a precipitate in this buffer, heat thebuffer at 37°C for 15 minutes and mix the bottle by gentle inversion.C. Plasmid Isolation Protocol1. Mix your overnight culture of cells completely and pipette 1.5 mL of cells into amicrocentrifuge tube. Centrifuge the culture in the microcentrifuge for 30 seconds. Discardthe supernatant.4