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During the plasmid miniprep we are going to grow cultures of E.coli cells, lyse them, and isolate plasmid DNA. Plasmid miniprep protocol: • Prepare numbered cell culture tubes with 4 ml of liquid LB medium and appropriate antibiotic • Pick colonies from a plate and inoculate media in the numbered tubes • Incubate the culture tubes in a shaker at 37 oC overnight • On the next day, save 1 ml of culture in a separate Eppendorf tube (for clone library) • Spin down the remaining 3 ml of culture and proceed with plasmid miniprep kit The only task for Day 2 is to start the miniprep cultures. Here I prepared three cell culture tubes with 4 ml of liquid LB medium with ampicillin in each of them. Right now I am going to inoculate the colonies from the plates into the tubes. I open tube one, pick a colony with a pipette tip and inoculate it into the tube. I repeat the same procedure for the other two tubes. The culture tubes are incubated overnight in a 37 oC shaker. We are now on the Day 3 of our cloning protocol. Right here on the bench I have the overnight cultures of E.coli which I am going to use for plasmid miniprep. But first, I am going to take 1 ml out of each of these culture tubes and save it in a separate Eppendorf tube. Later today, after I verify the clones by restriction digest, I will freeze these cultures in my Frozen Clone Collection. Now I am going to proceed with a plasmid miniprep kit to isolate DNA from these cultures. If you used the plasmid miniprep kits before, fast forward to the next step. First, I pellet the cells by spinning the culture tubes in a centrifuge. Here I have the pelleted cultures and now I am going to use standard miniprep kit (in this case it is an AccuPrep Plasmid MiniPrep kit from Bioneer), which consists of several buffers and columns, to isolate the plasmids. I start by discarding the supernatant from each of the tubes. Then I add 250 µl of a resuspension buffer. Then I resuspend the pelleted cells and transfer to new Eppendorf tubes. Now I am going to add 250 µl of a lysis buffer and mix the solution by gently flipping over the tubes. Then I add 350 µl of neutralization buffer to each of the tubes and again mix the solutions by gently flipping over the tubes. You can see that there is a cottage cheese-like formation in the tubes, which we are going to separate by spinning the tube for 10 minutes in a microcentrifuge. Then I transfer the supernatants to spin columns, spin 1 minute in a centrifuge, discard the flow through and add a wash buffer with ethanol. I spin it for 1 minute in a centrifuge again, discard the ethanol, and spin one more time.