Simply Cloning - Chapter 8 - Plasmid Miniprep

Описание: 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.