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| | <nav class="droptext arrows"> | | <nav class="droptext arrows"> |
| | <header class="hull"> | | <header class="hull"> |
| − | <label for="acc-close" class="hull-title">Basic Protocols</label> | + | <label for="acc-close" class="hull-title">Due to the simple nature of our dCas13a protein construct, it can be utilized and ‘personalized’ for a |
| | + | variety of purposes. Our main objectives range from ‘near’ future, to ‘future’.</label> |
| | </header> | | </header> |
| | <input type="radio" name="droptext" id="cb1" /> | | <input type="radio" name="droptext" id="cb1" /> |
| | <section class="hull"> | | <section class="hull"> |
| − | <label class="hull-title" for="cb1">Production of Lysogeny broth (LB)</label> | + | <label class="hull-title" for="cb1">Near Future</label> |
| | <label class="hull-close" for="acc-close"></label> | | <label class="hull-close" for="acc-close"></label> |
| − | <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 | + | <div class="hull-content">Although the processes within the central dogma have been studied and thoroughly characterized, |
| − | litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders
| + | recent studies suggest mRNA localization is a way of controlling and regulating protein production. ¹ This |
| − | with the water, we avoided shaking the glass bottle as it would cause froth and waste some of the
| + | mechanism appears to be similar to an induced suppressor that binds to DNA, thereby preventing its |
| − | LB.
| + | transcription, except there are significantly more (regulating) factors involved. Unfortunately, our |
| | + | knowledge is very limited as mRNA’s dynamic nature makes it significantly more difficult to study. |
| | + | As our protein, LuCAS, has the ability to attach to, and track any RNA, given it has its complementary |
| | + | crRNA sequence, the main objective is to track and investigate mRNA localization. LuCAS is sensitive and |
| | + | selective to singular basepairs, making it a reliable tracking tool without altering the cell and its |
| | + | intracellular function itself. |
| | <br> | | <br> |
| | When making the LB we also made another litre batch and added 15g of agar extract to be able to | | When making the LB we also made another litre batch and added 15g of agar extract to be able to |
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| | <input type="radio" name="droptext" id="cb2" /> | | <input type="radio" name="droptext" id="cb2" /> |
| | <section class="hull"> | | <section class="hull"> |
| − | <label class="hull-title" for="cb2">Production of SOB medium and magnesium stock</label> | + | <label class="hull-title" for="cb2">Future</label> |
| | <label class="hull-close" for="acc-close"></label> | | <label class="hull-close" for="acc-close"></label> |
| − | <div class="hull-content">Bringing together 20g of tryptone, 5g of yeast extract, 0.584g of NaCl, 0.186g of KCl and mixing it | + | <div class="hull-content">In a variety of disease states, mRNA could be used as a ‘biomarker’ and therapeutic target. Oncogenes |
| − | with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave
| + | for example, such as Epidermal growth factor receptor (EGFR), are often overexpressed early in cancers |
| − | to sterilise it, after it was taken out and let for it to cool down to below 60 o C. | + | and treatment courses based on the type of mutations causing the cancer have been shown to be |
| − | <br>
| + | crucial to treatment success; in this case for example, tyrosine kinase inhibitors or monoclonal |
| − | 10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm
| + | antibodies. Tools like LuCAS could potentially quantify and elucidate hyperproliferation and even qualify |
| − | filter sterilize was then used</div>
| + | mRNA as therapeutic target to halt hyper proliferation of key ‘driver’ genes by targeting their mRNA. |
| − | </section>
| + | Furthermore, monitoring mRNA expression in patients suffering chronic viral infections, such as HIV or |
| − | <input type="radio" name="droptext" id="cb3" />
| + | hepatitis B, can help pinpoint disease progression and aid in treatment path selection, as disease |
| − | <section class="hull">
| + | progression varies individually. HIV genes can be classified in early and late expression, with their |
| − | <label class="hull-title" for="cb3">Production of SOC medium and glucose stock</label>
| + | detection of corresponding mRNA, via LuCAS, helping to narrow down the stage of disease. |
| − | <label class="hull-close" for="acc-close"></label>
| + | The possible applications of LuCAS are many, as we simply propose a reliable and selective way to target |
| − | <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
| + | mRNA in vivo without altering cellular functions; be it for research, diagnostics or therapeutics. |
| − | bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in
| + | ~ LuCas is still a toddler and these are his first baby steps, we can’t wait to see him prosper and grow |
| − | to autoclave.
| + | into a young man that will change the world for the better</div> |
| − | <br>
| + | |
| − | 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.</div> | + | |
| − | </section>
| + | |
| − | <input type="radio" name="droptext" id="acc-close" />
| + | |
| − | <input type="radio" name="droptext" id="cb4" />
| + | |
| − | <section class="hull">
| + | |
| − | <label class="hull-title" for="cb4">Production of Glycerol stock</label>
| + | |
| − | <label class="hull-close" for="acc-close"></label>
| + | |
| − | <div class="hull-content">If you wish to store bacteria long term, you will need to create a Glycerol Stock after
| + | |
| − | inoculating an overnight liquid culture
| + | |
| − | <br>
| + | |
| − | <ul><li>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</li>
| + | |
| − | <li>The glycerol stock should then be frozen at -80 o C<ul>
| + | |
| − | <li> Successive freeze and thaw cycles will reduce the stocks shelf life</li></ul>
| + | |
| − | </li></ul></div>
| + | |
| | </section> | | </section> |
| | + | |
| | <input type="radio" name="droptext" id="acc-close" /> | | <input type="radio" name="droptext" id="acc-close" /> |
| − | <input type="radio" name="droptext" id="cb5" /> | + | <input type="radio" name="droptext" id="cb3" /> |
| | <section class="hull"> | | <section class="hull"> |
| − | <label class="hull-title" for="cb5">Running Agarose Gel</label> | + | <label class="hull-title" for="cb3">References</label> |
| | <label class="hull-close" for="acc-close"></label> | | <label class="hull-close" for="acc-close"></label> |
| − | <div class="hull-content">After the cells have been miniprepped and the plasmid put through a restriction digest, the agarose gel can be run. | + | <div class="hull-content">Kejiou, N. S. and Palazzo, A. F. (2017), mRNA localization as a rheostat to regulate subcellular gene |
| − | <br>
| + | expression. WIREs RNA, 8: n/a, e1416. doi:10.1002/wrna.1416</div> |
| − | <ul><li>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.</li>
| + | |
| − | <li>Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)</li>
| + | |
| − | <li>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)</li> | + | |
| − | <li>Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
| + | |
| − | solidify (maximum 30 mins)</li>
| + | |
| − | <li>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.</li>
| + | |
| − | <li>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!)</li>
| + | |
| − | <li>Load all of your digests into the wells 2,3, and 4.</li>
| + | |
| − | <li>Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps
| + | |
| − | don’t matter.</li>
| + | |
| − | <li>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.</li></ul></div>
| + | |
| | </section> | | </section> |
| | + | |
| | <input type="radio" name="droptext" id="acc-close" /> | | <input type="radio" name="droptext" id="acc-close" /> |
| | </nav> | | </nav> |
Future Ideas
