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| − | <nav class="droptext arrows">
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| − | <header class="hull">
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| − | <label for="acc-close" class="hull-title">Basic Protocols</label>
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| − | </header>
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| − | <input type="radio" name="droptext" id="cb1" />
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| − | <section class="hull">
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| − | <label class="hull-title" for="cb1">Production of Lysogeny broth (LB)</label>
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| − | <label class="hull-close" for="acc-close"></label>
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| − | <div class="hull-content">For 1 litre of LB a mixture of 10g of sodium chloride, 10g peptone, 5g of yeast extract as well as 1
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| − | litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders
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| − | with the water, we avoided shaking the glass bottle as it would cause froth and waste some of the
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| − | LB.
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| − | <br>
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| − | When making the LB we also made another litre batch and added 15g of agar extract to be able to
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| − | grow bacteria on plates.</div>
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| − | </section>
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| − | <input type="radio" name="droptext" id="cb2" />
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| − | <section class="hull">
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| − | <label class="hull-title" for="cb2">Production of SOB medium and magnesium stock</label>
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| − | <label class="hull-close" for="acc-close"></label>
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| − | <div class="hull-content">Bringing together 20g of tryptone, 5g of yeast extract, 0.584g of NaCl, 0.186g of KCl and mixing it
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| − | with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave
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| − | to sterilise it, after it was taken out and let for it to cool down to below 60 o C.
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| − | <br>
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| − | 10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm
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| − | filter sterilize was then used</div>
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| − | </section>
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| − | <input type="radio" name="droptext" id="cb3" />
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| − | <section class="hull">
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| − | <label class="hull-title" for="cb3">Production of SOC medium and glucose stock</label>
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| − | <label class="hull-close" for="acc-close"></label>
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| − | <div class="hull-content">Once again bring 20g of tryptone, 5g of yeast of extract, 0.584g of NaCl, 0.186g of KCL, and then
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| − | bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in
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| − | to autoclave.
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| − | <br>
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| − | 10ml of 2M Mg 2+ stock and then bring it to 100ml with milllipure water, filter sterilize it with 0.2m
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| − | and then final add 20ml of 1M glucose stock.</div>
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| − | </section>
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| − | <input type="radio" name="droptext" id="acc-close" />
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| − | <input type="radio" name="droptext" id="cb4" />
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| − | <section class="hull">
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| − | <label class="hull-title" for="cb4">Production of Glycerol stock</label>
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| − | <label class="hull-close" for="acc-close"></label>
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| − | <div class="hull-content">If you wish to store bacteria long term, you will need to create a Glycerol Stock after
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| − | inoculating an overnight liquid culture
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| − | <br>
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| − | <ul><li>Once bacterial growth has been achieved, 500μL of the overnight liquid
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| − | culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
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| − | should be gently mixed</li>
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| − | <li>The glycerol stock should then be frozen at -80 o C<ul>
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| − | <li> Successive freeze and thaw cycles will reduce the stocks shelf life</li></ul>
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| − | </li></ul></div>
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| − | </section>
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| − | <input type="radio" name="droptext" id="acc-close" />
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| − | <input type="radio" name="droptext" id="cb5" />
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| − | <section class="hull">
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| − | <label class="hull-title" for="cb5">Running Agarose Gel</label>
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| − | <label class="hull-close" for="acc-close"></label>
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| − | <div class="hull-content">After the cells have been miniprepped and the plasmid put through a restriction digest, the agarose gel can be run.
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| − | <br>
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| − | <ul><li>Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a
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| − | 250ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20
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| − | seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too
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| − | much. Make up the evaporated volume to 50ml with distilled water.</li>
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| − | <li>Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)</li>
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| − | <li>Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use
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| − | a p1000 pipette set to 1ml. Let it dry (about 5 mins max)</li>
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| − | <li>Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
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| − | solidify (maximum 30 mins)</li>
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| − | <li>When the gel has set, remove the comb from the tank (gently!) and then cover the whole
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| − | tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.</li>
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| − | <li>Now, the samples need to be loaded. Load some DNA markers (ask technical services for a
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| − | tube of this and load the whole tube) into well 1( left hand side) and then choose what you
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| − | load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)</li>
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| − | <li>Load all of your digests into the wells 2,3, and 4.</li>
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| − | <li>Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps
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| − | don’t matter.</li>
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| − | <li>Once the visible markers have reached the half way point of the tank, turn off the power
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| − | supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a
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| − | UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.</li></ul></div>
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| − | </section>
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| − | <input type="radio" name="droptext" id="acc-close" />
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| − | </nav>
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Attribution
