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.