Difference between revisions of "Team:Lethbridge HS/Methods"

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             <h1 id="firsttitle" style=""> <b>PROTOTYPE</b></h1>
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             <h1 id="firsttitle" style=""> <b>METHODS</b></h1>
 
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<li><a href="#first">Our Goal</a></li>
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<li><a href="#first">Plasmid Resuspension l</a></li>
<li><a href="#first">Initial Design</a></li>
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<li><a href="#first">Transformation</a></li>
<li><a href="#first">Buying All Of The Parts</a></li>
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<li><a href="#first">DNA Quantification</a></li>
<li><a href="#first">Putting Our Prototype Together</a></li>
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<li><a href="#first">LB Media</a></li>
<li><a href="#first">Results</a></li>
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<li><a href="#first">Overnight Culture (Colony pick)</a></li>
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<li><a href="#first">Restriction Digest</a></li>
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<li><a href="#first">Ligation </a></li>
 
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<h3>Our Goal</h3>
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<h3>Plasmid Resuspension l</h3>
 
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<p>For our prototype we wanted to really showcase how our construct would work in a real life scenario in terms of forming an actual clot and how long it would take as well as the application method of using a pre-filled syringe.</p>
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<p>Resuspend ordered plasmid (construct) in a 0.1mM Tris, 0.1uM EDTA buffer solution. Ensure circularization with transformation.</p>
<h3>Initial Design</h3>
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<h3>Transformation</h3>
<p>For our initial design of our prototype we started off with the blood being contained in a tank where it will then be sucked up and go through some medical tubing. Then there would be a flow meter that would measure the pressure of the blood. The tubing will lead the blood to our slab of meat that is contained inside of our splash box. (The splash box initial design would be made out of plexiglass so that the viewer can see nicely through it). At this point the blood will be gushing out, simulating an actual wound site. Then more medical tubing will allow for the excess blood to go through our second flow meter where it will again measure the pressure before it is collected in our blood bucket at the end.</p>
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<p>Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature. Mix 5 μL of DNA into 20 μL of competent cells in a micro centrifuge tube. Incubate the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation tube by placing the tube into a 42°C water bath for 45 seconds. Put the tubes back on ice for 2 min. Add 250 μL LB to the bacteria and grow in 37°C shaking incubator for an hour. Plate 200 uL and 50 uL on 2 separate plates. Incubate plates at 37°C overnight.</p>
<h3>Buying All Of The Parts</h3>
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<h3>DNA Quantification </h3>
 
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<p> When it came to actually buying all of our prototype parts, we had to take the prices of every part into perspective. Because the flow meters are really expensive we decided not to include them in the final design of our prototype. We bought four different types of medical tubing (one clear type of tubing that had a fairly small diameter, another clear one with a larger diameter, a yellow flexible one that also had a large diameter and a blue one that had a small diameter). In total the tubing was around $30.00 and the one that we chose to use in our prototype was the small clear one because we want to show the blood as it travels through it and it fit around the tip of the syringe nicely. We decided to just use a syringe instead of using the flow meters to pump the blood up from the container, so we will fill the syringe up with the blood before we attach it to the tubing. We also bought a plastic clear container that was $17.00 instead of cutting and assembling the plexiglass into a box.<p/>
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<p>Using a NanoDrop, place an initial blank of d2H2O. Remove the water then add a DNA sample then use the 260/280nm nucleic acid measurement tool to quantify DNA concentration. <p/>
<h3>Putting Our Prototype Together</h3>
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<h3>LB Media</h3>
<p>For the assembly of our prototype we started off by drilling a hole in one of the sides and then we drilled another hole opposite of the first one so the tubing will go through the splash box. These holes were made into the shorter sides of the rectangular box. We cut the middle part of one of the longer sides so that the viewers can get a better view of the steak and our product being applied. We will fill the syringe up with blood from the blood container, then attach the medical tubing to the tip of the syringe. The medical tubing goes through the splash box and into our steak, then more medical tubing is attached to the other side of the steak and then out. At the end of the tubing there will be another container to collect the blood. Because we are not using a flow meter in our prototype to measure the pressure of the blood, we can qualitatively measure it by looking at the video that we took of our prototype in action, frame by frame.</p>
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<p> Add 10g of Tryptone, 10g of NaCl, 5g of Yeast Extract to just under 1L of d2H2O. Take the solution and obtain a pH of 7.5. Autoclave to obtain LB media or add 5g of agar to 500 mL then autoclave to obtain agar plates. </p>
<h3> Results</h3>
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<h3>Overnight Culture (Colony pick)</h3>
<p>We have only done test runs of water and blood through our prototype, we haven’t tested our construct with it yet but we are planning to do that in the future. When we tested our prototype with water, we just filled a syringe full of water and attached it to the medical tubing that ran through our splash box. This was to see if the design of our prototype was successful or not. We ran it with the blood in our lab, for this we made a small incision in the medical tubing that was inside of our splash box. This simulated a wound site and this was successful as blood did gush out of the cut.</p>
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<p>Identify a colony on an LB + Agar plate and with a small pipette tip place the tip into the colony then removing it without damaging the agar in and around the colony. Place the pipet tip into a 5 mL LB media tube. If needed add appropriate antibiotic to the solution. Amp was used for our construct. Incubate tubes at 37°C overnight but ensure that it doesn’t stay too long or the antibiotic can become deactivated leading to contamination.</p>
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<h3>Restriction Digest</h3>
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<p> In a 1.5 mL tube add the following components and make alterations based on DNA concentrations so that the tube can have a final volume of 30 uL. Add approximately 1 ug of DNA, 1 uL of each Restriction Enzyme, 3 uL of 10x Cut buffer. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Then if using this DNA right away deactivate the enzymes by bringing the temperature up to 70°C for 15 minutes or longer.</p>
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<h3>Ligation </h3>
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<p>Combine the following in a PCR or small micro centrifuge tube: 25ng Vector DNA, 75ng Insert DNA, Ligase Buffer, 1μL T4 DNA Ligase and d2H2O to total it to 10 or 15 uL. Incubate at room temperature for 2hr, or overnight. Proceed with bacterial transformation.</p>
 
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Revision as of 02:38, 20 October 2016

Lethbridge HS iGEM 2016

METHODS

Plasmid Resuspension l

Resuspend ordered plasmid (construct) in a 0.1mM Tris, 0.1uM EDTA buffer solution. Ensure circularization with transformation.

Transformation

Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature. Mix 5 μL of DNA into 20 μL of competent cells in a micro centrifuge tube. Incubate the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation tube by placing the tube into a 42°C water bath for 45 seconds. Put the tubes back on ice for 2 min. Add 250 μL LB to the bacteria and grow in 37°C shaking incubator for an hour. Plate 200 uL and 50 uL on 2 separate plates. Incubate plates at 37°C overnight.

DNA Quantification

Using a NanoDrop, place an initial blank of d2H2O. Remove the water then add a DNA sample then use the 260/280nm nucleic acid measurement tool to quantify DNA concentration.

LB Media

Add 10g of Tryptone, 10g of NaCl, 5g of Yeast Extract to just under 1L of d2H2O. Take the solution and obtain a pH of 7.5. Autoclave to obtain LB media or add 5g of agar to 500 mL then autoclave to obtain agar plates.

Overnight Culture (Colony pick)

Identify a colony on an LB + Agar plate and with a small pipette tip place the tip into the colony then removing it without damaging the agar in and around the colony. Place the pipet tip into a 5 mL LB media tube. If needed add appropriate antibiotic to the solution. Amp was used for our construct. Incubate tubes at 37°C overnight but ensure that it doesn’t stay too long or the antibiotic can become deactivated leading to contamination.

Restriction Digest

In a 1.5 mL tube add the following components and make alterations based on DNA concentrations so that the tube can have a final volume of 30 uL. Add approximately 1 ug of DNA, 1 uL of each Restriction Enzyme, 3 uL of 10x Cut buffer. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Then if using this DNA right away deactivate the enzymes by bringing the temperature up to 70°C for 15 minutes or longer.

Ligation

Combine the following in a PCR or small micro centrifuge tube: 25ng Vector DNA, 75ng Insert DNA, Ligase Buffer, 1μL T4 DNA Ligase and d2H2O to total it to 10 or 15 uL. Incubate at room temperature for 2hr, or overnight. Proceed with bacterial transformation.