Simply Cloning - Chapter 8 - Plasmid Miniprep

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.

[@dubie8634]

great!

[@CloningStrategies]

When supernatant goes through a column, plasmid DNA binds to resin and stays there. The column with DNA is washed with ethanol solution to get remove salts. The centrifuging steps are to wash the column with ethanol and then to get rid of that ethanol. Cheers! :)

[@CloningStrategies]

Do you mean at 4:20? After addition of neutralization buffer, when "cottage-cheese-like" precipitate forms (with all the proteins and polysaccharides), this precipitate has to be separated from the rest of the solution that contains DNA

[@haafizah786]

Why do centrifuge again after we discard the supernatant????...

[@hitendrasingh850]

thank u sir but i was also used miniprep kit for isolation but did not seen band in pcr amplified dna what is the reson....?

[@haafizah786]

@haafizah786 sorry I meant why do we centrifuge the supernatant again?

[@kailynthompson1305]

Hello, I was wondering at what speed you used the centrifuge each time, I couldn't make it out in the video, and for how long?

[@CloningStrategies]

1 min at maximum speed - 13 000 rpm on that centrifuge

[@babamitzsam]

How do you create a miniprep kit?

[@CloningStrategies]

@babamitzsam: You buy them.