Difference between revisions of "Team:Kent/HP/Silver"

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<label for="acc-close" class="hull-title">Basic Protocols</label>
 
<label for="acc-close" class="hull-title">Basic Protocols</label>
 
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<label class="hull-title" for="cb1">Production of Lysogeny broth (LB)</label>
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<span>London Meetup</span>
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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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<p>Our team has strived to make human practices the solid foundation of our project right from the start. We wanted to ensure we fully understood the implications of any project we chose to pursue while addressing current global problems.  
 
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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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We held a brainstorming session where we bounced ideas off of one another for potential project ideas and had narrowed it down to a top five. We decided we needed to consult with our immediate community to scope out what issues they deemed important that needed immediate addressing. Through different outreach opportunities, we were able to comprehend the controversy surrounding GMOs and genetic engineering in general. This caused us to critically analyze our project ideas before deciding upon ‘LuCAS’: a novel way of mRNA localization using CRISPR - dCas13a.
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grow bacteria on plates.</div>
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<label class="hull-title" for="cb2">Production of SOB medium and magnesium stock</label>
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<span>Judd High School</span>
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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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<p>This year, three members of our team: Laulwa Al Salloum, Laurens Heling and Dan Brunkow, were able to work alongside Team Judd_UK in the InterLab study. They were able to use our lab and equipment to obtain the necessary readings for their InterLab while we mentored two of their members; Nikita Shukan and Mateo Hoare through the process, as well as aiding them in their managing of the different aspects of the study, such as: using the Gilson pipettes, using the plate reader, incubating the samples, etc. In return, they generously donated their InterLab DNA from Distribution kit plate 6 for us to transform as we were unsuccessful in transforming the DNA from our own kit into the E.coli DH5 alpha cells.
 
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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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Being able to work alongside another iGEM team was incredibly useful in viewing the study from a different point of view as Judd_UK were experiencing all the lab work for the first time as they are a High School team. Being able to mentor them allowed us to work through the study step by step until it became second nature to perform all the readings.
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filter sterilize was then used</div>
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<label class="hull-title" for="cb3">Production of SOC medium and glucose stock</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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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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Link to Judd’s wiki on our collaboration: https://2017.igem.org/Team:Judd_UK/Collaborations
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and then final add 20ml of 1M glucose stock.</div>
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<img src="https://static.igem.org/mediawiki/2017/9/9e/T--Kent--Collaboration3.png">
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<label class="hull-title" for="cb4">Production of Glycerol stock</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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<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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<label class="hull-title" for="cb5">Running Agarose Gel</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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<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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Revision as of 02:19, 16 December 2017


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