Exeter/8 July 2013

From 2013.igem.org

Extracting our plasmids from our transformed cells

The MiniPrep kit needs 35ml ethanol added to the "Wash Solution" and RNAase 2 added to the "Resuspension Solution", which then needs storing 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 250ul 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 250ul lysis solution and invert 4-6 times.

- Add 350ul 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 500ul 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 50ul 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 -20oC 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...

- 200ul liquid cell culture

- 200ul 60% glycerol stock

Stored at -20oC.

Digests of our transformed cells' plasmids

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

Method for digest:

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

- Add 100ml TBE buffer and microwave until bubbling.

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

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

Setting up the digest:

We need...

- 120ul distilled water

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

- 5ul Enzyme 1 (XbaI)

- 5ul Enzyme 2 (SpeI)

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

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

Incubate the tubes at 37oC for 30 minutes using a waterbath

TO BE CONTINUED!