Exeter/8 July 2013

MiniPrep plasmid extraction

Extracting our plasmids from our transformed cells

The MiniPrep kit needs 35 ml ethanol added to the "Wash Solution" and RNAase 2 added to the "Resuspension Solution", which then needs to be stored in the fridge.

Method:

- Pipette 1ml of liquid growth culture into each of 3 Eppendorfs (same liquid culture for each).

- Centrifuge at 8,000 rpm for 5 minutes and pour off the supernatant.

- Add 250 µl resuspension solution to one of the Eppendorfs. Resuspend the pellet, then draw up the contents of this Eppendorf and pipette into the next Eppendorf. Resuspend the pellet in this Eppendorf, then draw up the contents and pipette into the third and final Eppendorf. Resuspend the pellet in this Eppendorf.

- This last Eppendorf is what we use for the rest of the MiniPrep. This is to get the maximum amount of plasmid as possible from the transformed cells.

- Add 250 µl lysis solution and invert 4-6 times.

- Add 350 µl neutralisation solution and invert 4-6 times.

- Centrifuge at 13,400 rpm for 5 minutes.

- Pour (not pipette!) the supernatant into the separating column and centrifuge the column for 1 minute at 13,400 rpm.

- Pour off the run-through.

- Add 500 µl wash solution and centrifuge at 13,400 rpm for 1 minute. Discard run-through. Repeat this step (washing column twice overall).

- Centrifuge empty column at 13,400 rpm for 1 minute.

- Transfer the column into an Eppendorf and add 50 µl elution buffer.

- Rest the column for 2 minutes, then centrifuge for 2 minutes at 13,400 rpm. DO NOT THROW THE SUPERNATANT AWAY! This contains our plasmids, which can now be stored at -20 °C until required.

Glycerol stocks

The glycerol stops the cell walls being broken by formation of ice crystals, so we can freeze our cells.

Each Eppendorf contains...

- 200 µl liquid cell culture

- 200 µl 60% glycerol stock

Stored at -20°C.

Digests of our transformed cells' plasmids

We are using a premade TBE buffer, which has to be diluted down. 100 ml of TBE buffer concentrate is mixed with 900 ml of distilled water.

Method for digest:

- Weigh 1 g agarose powder into a 250 ml conical flask.

- Add 100 ml TBE buffer and microwave until bubbling.

- Add 5 µl midori green and swirl (alternatively, 2.5 ul of etidium bromide could be added, but this is toxic, so we've chosen to avoid it).

- Pour into a prepared tray and add the comb, which will make our wells.

Setting up the digest:

We need...

- 120 µl distilled water

- 20 µl buffer (ours includes a dye, so we can see where our DNA is up to on the gel).

- 5 µl Enzyme 1 (XbaI)

- 5 µl Enzyme 2 (SpeI)

Vortex until mixed. Pipette 15 µl of this "master mix" into a PCR tube.

To each tube, add 5 µl of DNA and mix.

Incubate the tubes at 37 °C for 30 minutes using a waterbath

In the gel, add 10 µl of chosen DNA ladder to the first well. Add 20 µl of each fraction of DNA you want to run to the subsequent wells.

Run the gel for 50 minutes on 120 Watts.

View the gel under UV light.

Take me back to the notebook.