Team:Kent/HP/Gold Integrated

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Gold

Basic Protocols

Production of Lysogeny broth (LB)

For 1 litre of LB a mixture of 10g of sodium chloride, 10g peptone, 5g of yeast extract as well as 1 litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders with the water, we avoided shaking the glass bottle as it would cause froth and waste some of the LB.
When making the LB we also made another litre batch and added 15g of agar extract to be able to grow bacteria on plates.

Production of SOB medium and magnesium stock

Bringing together 20g of tryptone, 5g of yeast extract, 0.584g of NaCl, 0.186g of KCl and mixing it with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave to sterilise it, after it was taken out and let for it to cool down to below 60 o C.
10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm filter sterilize was then used

Production of SOC medium and glucose stock

Once again bring 20g of tryptone, 5g of yeast of extract, 0.584g of NaCl, 0.186g of KCL, and then bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in to autoclave.
10ml of 2M Mg 2+ stock and then bring it to 100ml with milllipure water, filter sterilize it with 0.2m and then final add 20ml of 1M glucose stock.

Production of Glycerol stock

If you wish to store bacteria long term, you will need to create a Glycerol Stock after inoculating an overnight liquid culture

• Once bacterial growth has been achieved, 500μL of the overnight liquid culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it should be gently mixed
• The glycerol stock should then be frozen at -80 o C
  • Successive freeze and thaw cycles will reduce the stocks shelf life

Running Agarose Gel

After the cells have been miniprepped and the plasmid put through a restriction digest, the agarose gel can be run.

• Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a 250ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20 seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too much. Make up the evaporated volume to 50ml with distilled water.
• Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)
• Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use a p1000 pipette set to 1ml. Let it dry (about 5 mins max)
• Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and solidify (maximum 30 mins)
• When the gel has set, remove the comb from the tank (gently!) and then cover the whole tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.
• Now, the samples need to be loaded. Load some DNA markers (ask technical services for a tube of this and load the whole tube) into well 1( left hand side) and then choose what you load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)
• Load all of your digests into the wells 2,3, and 4.
• Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps don’t matter.
• Once the visible markers have reached the half way point of the tank, turn off the power supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.