{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page_content %}
<br>
<div class="model-heading">
<h1 id="experiment-heading">
Experimental Design
</h1>
</div>
<div class="Content" style="max-height: 100%;">
<img src="https://static.igem.wiki/teams/4931/wiki/ed1.png" class="experImg" alt="First Image for ED page">
<br><br>
<img src="https://static.igem.wiki/teams/4931/wiki/ed2.jpg" class="experImg" alt="Second image of ED page">
<br><br>
<img src="https://static.igem.wiki/teams/4931/wiki/ed3.jpg" class="experImg" alt="Third Image for ED page">
<p>Plasmid vector being used : <b>pNZ8148</b></p>
<div class="model-heading">
<h1 id="experiment-heading">
Protocols
</h1>
</div>
<br><br>
<div class="model-subheading">
<h2>PCR</h2>
</div>
<br><br>
<img src="https://static.igem.wiki/teams/4931/wiki/ed4.png" class="experImg" alt="Third Image for ED page">
<br><br>
<p>
<b>Intermediate steps repeated for 35 cycles</b>
<br><br>
<b>Mix:</b>
</p>
<ul type="disc">
<li class="list-element">Template solution - 10uL</li>
<li class="list-element">FWD primer - 2.5uL</li>
<li class="list-element">Rev primer - 2.5uL</li>
<li class="list-element">HF buffer - 10uL</li>
<li class="list-element">dNTPs - 1uL</li>
<li class="list-element">Polymerase - 0.5uL</li>
<li class="list-element">dH2O - 23.5 uL</li>
</ul>
<p><b>Total - 50uL</b></p>
<br><br>
<div class="model-subheading">
<h2>Golden Gate Assembly</h2>
</div>
<br><br>
<ul type="disc">
<li class="list-element">Fragment + dH20 -> 16uL</li>
<li class="list-element">Bsa1-HF -> 0.5uL</li>
<li class="list-element">T4 Ligase -> 0.5uL</li>
<li class="list-element">T4 Ligase Buffer - 2uL</li>
</ul>
<p><b>Run :</b></p>
<ul type="disc">
<li class="list-element">37C -> 5min</li>
<li class="list-element">37C -> 5min</li>
<li class="list-element">16C -> 5min</li>
<li class="list-element">Repeat above 2 steps for 30 cycles</li>
<li class="list-element">60C -> 15min</li>
</ul>
<br><br>
<div class="model-subheading">
<h2>Gel Extraction: QIAquick® Gel Extraction Kit</h2>
</div>
<br><br>
<ol type="1" class="ed-list">
<li class="list-element">Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li>
<li class="list-element">Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100
mg gel ~100 μl). Note: For >2% agarose gels, add 6 volumes of Buffer QG.</li>
<li class="list-element">Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the
tube every 2–3 min to help dissolve the gel. After the gel slice has dissolved completely, check that the color of
the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or
violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.</li>
<li class="list-element">Add 1 gel volume of isopropanol to the sample and mix.</li>
<li class="list-element">Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold
until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into
the same tube. For sample volumes >800 μl, load and spin/apply vacuum again.</li>
<li class="list-element">If DNA will subsequently be used for sequencing, <i>in vitro</i> transcription, or microinjection,
add 500 μl of Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and
place the QIAquick column back into the same tube.</li>
<li class="list-element">To wash, add 750 μl of Buffer PE to the QIAquick column and centrifuge for 1 min or apply
vacuum. Discard flow-through and place the QIAquick column back into the same tube.</li>
<li class="list-element">Place the QIAquick column into a clean 1.5 ml microcentrifuge tube.</li>
<li class="list-element">To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the
QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl of Buffer EB to
the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the
addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the
yield of purified DNA.</li>
</ol>
<br><br>
<div class="model-subheading">
<h2>Plasmid Isolation: QIAprep® Spin Miniprep Kit</h2>
</div>
<br><br>
<ol type="1" class="ed-list">
<li class="list-element">Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge
tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the
pellet. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure
LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or
pipetting up and down until no cell clumps remain.</li>
<li class="list-element">Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by
inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and contamination of
plasmid. Continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis
reaction to proceed for more than 5 min. If LyseBlue has been added to Buffer P1, the cell suspension will turn
blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension.</li>
<li class="list-element">Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times. To
avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture
volumes (e.g., ≥5 ml) may require inverting up to 10 times. The solution should become cloudy. If LyseBlue reagent
has been used, the suspension should be mixed until all trace of blue is gone and the suspension is colorless.
</li>
<li class="list-element">Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact
white pellet will form.</li>
<li class="list-element">Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting.
</li>
<li class="list-element">Centrifuge for 30–60 s. Discard the flow through.</li>
<li class="list-element">Recommended: Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and centrifuging
for 30–60 s. Discard the flow through. This step is necessary to remove trace nuclease activity when using endA+
strains, such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of
nuclease activity or high carbohydrate content. Host strains, such as XL-1 Blue and DH5α, do not require this
additional wash step.</li>
<li class="list-element">Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.</li>
<li class="list-element">Discard the flow through, and centrifuge at full speed for an additional 1 min to remove
residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow through is
discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic
reactions.</li>
<li class="list-element">Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add
50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1
min, and centrifuge for 1 min.</li>
</ol>
<br><br>
</div>
<div class="model-subheading">
<h2>Fluorescence Assay</h2>
</div>
<br><br>
<ul type="disc" class="ed-list">
<li class="list-element">Overnight cultures were used for creating 1/100 times diluted subcultures in biological
duplicates. The Lactococcus strains were grown at optimal conditions reported for them.</li>
<li class="list-element">These subcultures were grown till the end of their exponential phase. The samples were then
centrifuged. The media was discarded and the pellets were washed with NaCl (0.9%) solution.</li>
<li class="list-element">The pellets were re-homogenized in 0.9% NaCl and 100uL was taken from each sample (duplicate
sample measurement for each biological replicate) in a clear bottomed 96 well plate.</li>
<li class="list-element">Fluorescence/OD values were measured after subtraction of the negative control. The mean from
all the samples and biological replicates was used for the creation of the respective graph.</li>
</ul>
<br><br>
<div class="model-subheading">
<h2>Electroporation</h2>
</div>
<br><br>
<ul type="disc" class="ed-list">
<li class="list-element">Golden gate reaction mixture and electro-competent L.Lactis cells were mixed and added into
an electroporation cuvette (0.2cm) and allowed to homogenize for 20-30 minutes.</li>
<li class="list-element">A 2500 Volt pulse is passed through the cuvette and 1ml SGM17 media is added to the mixture
immediately for 5 minutes.</li>
<li class="list-element">The cultures are transferred to 1.5ml tubes and incubated for 1 hour.</li>
<li class="list-element">The cultures were spread plated on M17 Agar with appropriate antibiotics and incubated for 24
hours.</li>
<li class="list-element">The colonies were picked for overnight cultures with appropriate antibiotics and used for
creating glycerol stocks.</li>
</ul>
{% endblock %}