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protocols.html
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<body>
<div id="backgroundimage1"><div class="background-gradient-down"><p class="wide-heading-type mainwrap align-center">PROTOCOLS</p></div></div>
<div id="panel1"class="link-slideup">
<p class="body-type mainwrap"><strong>Below is an interactive list of the laboratory techniques employed by the team. Click to navigate to that protocol.</strong></p>
<p class="mainwrap body-type">
<a href="#KODPCR">KOD PCR</a>,
<a href="#ColonyPCR">Colony PCR</a>,
<a href="#OEPCR">Overlap Extention PCR</a>,
<a href="#1AGel">Making a 1% Agarose Gel</a>,
<a href="#Electrophoresis">Agarose Gel Electrophoresis</a>,
<a href="#CellStock">Cell Stock</a>,
<a href="#Miniprep">Miniprep</a>,
<a href="#GelExtraction">Gel Extraction</a>,
<a href="#PCRCleanup">PCR Cleanup</a>,
<a href="#Digestion">Digestion</a>,
<a href="#Ligation">Ligation</a>,
<a href="#Transformation">Transformation</a>,
<a href="#LiquidCultures">Liquid Cultures</a>,
<a href="#TestCuts">Test Cuts</a>,
<a href="#Gibson">Gibson</a>,
<a href="#Recombination">Recombination Reaction</a>,
<a href="#CellFree">Cell-Free Reaction</a>,
<a href="#MakingCF">Making Cell Free</a>
</p>
<div id="protocol-accordion"class="mainwrap">
<a class="anchor" name="KODPCR"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">KOD PCR</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>5% DMSO</li>
<li>2x KOD Hot Start Master Mix</li>
<li>PCR Tubes</li>
<li>Forward Primers(s) [10 uM]</li>
<li>Reverse Primer(s) [10 uM]</li>
<li>DNA Template</li>
<li>Deionized water</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 18.5 uL of deionized water to PCR tube</li>
<li>Add 1.5 µl of both the requisite Forward and Reverse Primers to PCR tube</li>
<li>Add 1 ul of DNA template</li>
<li>Add 27.5 µl of KOD Hot start Master Mix to PCR tube</li>
<li>Centrifuge for 10 seconds to remove air bubbles</li>
<li>Place in Thermocycler</li>
</ol>
<p class="body-type">
Thermocycler Conditions
</p>
<table class="body-type" frame="box">
<tr>
<th>Number of Cycles</th>
<td>From 20 - 40</td>
</tr>
<tr>
<th>Annealing Temperature</th>
<td>Set temperature to the lowest primer melt temperature</td>
</tr>
<tr>
<th>Extension Time</th>
<td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/kb</td>
</tr>
</table>
</div>
<a class="anchor" name="ColonyPCR"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Colony PCR</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>5% DMSO</li>
<li>2x KOD Hot Start Master Mix</li>
<li>PCR Tubes</li>
<li>Forward Primers(s) [10 uM]</li>
<li>Reverse Primer(s) [10 uM]</li>
<li>DNA Template</li>
<li>Deionized water</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Prepare the DNA template by picking a colony from a plate and mixing it into 10 uL of deionized water in a PCR tube</li>
<li>Add 17.5 µl of deionized water to another PCR tube</li>
<li>Add 1.5 µl of both the requisite Forward and Reverse Primers to the second PCR tube</li>
<li>Add 2 ul of water containing cells with DNA template sample</li>
<li>Add 27.5 µl of KOD Hot Start Master Mix</li>
<li>Centrifuge for 10 seconds to remove air bubbles</li>
<li>Place in Thermocycler</li>
</ol>
<p class="body-type">
Thermocycler Conditions
</p>
<table class="body-type" frame="box">
<tr>
<th>Number of Cycles</th>
<td>From 20 - 40</td>
</tr>
<tr>
<th>Annealing Temperature</th>
<td>Set temperature to the lowest primer melt temperature</td>
</tr>
<tr>
<th>Extension Time</th>
<td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/kb</td>
</tr>
</table>
</div>
<a class="anchor" name="OEPCR"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Overlap Extension PCR</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>5% DMSO</li>
<li>2x KOD Hot Start Master Mix</li>
<li>PCR Tubes</li>
<li>Forward Primers(s) [10 uM]</li>
<li>Reverse Primer(s) [10 uM]</li>
<li>DNA Template</li>
<li>Deionized water</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add equimolar concentrations of each DNA sample to a PCR tube</li>
<li>Add 1.5 uL of each Forward and Reverse Primer</li>
<li>Add 27.5 uL of KOD Hot Start Master Mix</li>
<li>Add deionized water until the total volume is 50 uL</li>
<li>Place in Themocycler</li>
</ol>
<p class="body-type">
Thermocycler Conditions
</p>
<table class="body-type" frame="box">
<tr>
<th>Number of Cycles</th>
<td>From 20 - 40</td>
</tr>
<tr>
<th>Annealing Temperature</th>
<td>Set temperature to the lowest primer melt temperature</td>
</tr>
<tr>
<th>Extension Time</th>
<td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/kb</td>
</tr>
</table>
</div>
<a class="anchor" name="1AGel"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Making a 1% Agarose Gel</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>Agarose</li>
<li>1x TAE Buffer</li>
<li>Ethidium Bromide</li>
<li>250 mL Flask</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 0.6 g of agarose to 250 mL flask</li>
<li>Add 60 mL of 1x TAE buffer and mix by swirling the flask</li>
<li>Microwave mixture for 60 seconds</li>
<li>Add 3 uL of Ethidium Bromide and mix gently</li>
<li>Pour into gel tray with the desired comb size and wait 30 minutes for it to solidify</li>
</ol>
</div>
<a class="anchor" name="Electrophoresis"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Agarose Gel Electrophoresis</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>Agarose Gel</li>
<li>6x Loading Dye</li>
<li>2 log DNA ladder</li>
<li>Gel Box</li>
<li>1x TAE Buffer</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 6x loading dye to each sample</li>
<li>Place agarose gel into gel box</li>
<li>Fill gel box with 1x TAE buffer to fill line</li>
<li>Load 2 log ladder and samples into wells</li>
<li>Run gel at 135 V for 30-40 minutes</li>
</ol>
</div>
<a class="anchor" name="CellStock"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Cell Stock</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>Liquid Cell Culture</li>
<li>50% Glycerol</li>
<li>Cryofreezer Tube</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 600 ul of glycerol into cryotube</li>
<li>Add 600 ul of culture to cryotube</li>
<li>Mix gently by pipetting up and down</li>
<li>Freeze and store at -80℃</li>
</ol>
</div>
<a class="anchor" name="Miniprep"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Miniprep</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>1.7 mL Microcentrifuge Tubes</li>
<li>Spin Columns</li>
<li>Spin Tubes</li>
<li>MX1 Buffer</li>
<li>MX2 Buffer</li>
<li>MX3 Buffer</li>
<li>WN Buffer</li>
<li>WS Buffer</li>
<li>Elution Buffer</li>
<li>Centrifuge</li>
<li>Vacuum Manifold</li>
<li>55℃ Bead Bath</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Prepare the Elution Buffer by placing it in the 55℃ bead bath</li>
<li>Transfer the sample to a microcentrifuge tube and spin for 2 minutes at 13,000 rpm</li>
<li>Pour off the supernatant from the pellet</li>
<li>Pipette 200 uL of MX1 into the samples and vortex to resuspend</li>
<li>Pipette 250 uL of MX2 into the samples and invert the tube ~5 times</li>
<li>Pipette 350 uL of MX3 into the samples and invert the tube ~5 times</li>
<li>Centrifuge at MAX speed for 5 minutes</li>
<li>Pour the supernatant off the microcentrifuge tubes into spin columns and turn on the vacuum manifold</li>
<li>Pipette 500 uL of WN buffer into each column</li>
<li>Pipette 700 uL of WS buffer into each column</li>
<li>Turn of vacuum manifold and place spin columns in collection tubes</li>
<li>Centrifuge for 3 minutes at MAX speed and discard collection tubes. Place the spin column in a new microcentrifuge tube</li>
<li>Pipette 50 uL of Elution Buffer right above the membrane of the column without touching the membrane with the pipette tip. Let the column sit for 2 minutes</li>
<li>Centrifuge for 60 seconds at MAX speed</li>
<li>Nanodrop DNA for concentration and store in at -20℃</li>
</ol>
</div>
<a class="anchor" name="GelExtraction"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Gel Extraction</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>1.7ml Microcentrifuge Tubes</li>
<li>Spin Columns</li>
<li>Spin Tubes</li>
<li>GEX Buffer</li>
<li>WN Buffer</li>
<li>WS Buffer</li>
<li>Centrifuge</li>
<li>Vacuum Manifold</li>
<li>55℃ Bead Bath</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Prepare the Elution Buffer by placing it in the 55℃ bead bath</li>
<li>Place excised gel fragment in a microcentrifuge tube and add 700 uL of GEX buffer</li>
<li>Place tubes in the 55℃ bead bath and wait for the gel fragment to fully dissolve</li>
<li>Transfer contents to spin columns that are place on the vacuum manifold. Turn on manifold</li>
<li>Pipette 500 uL of WN buffer into each column</li>
<li>Pipette 500 uL of WS buffer into each column</li>
<li>Turn of vacuum manifold and place spin columns in collection tubes</li>
<li>Centrifuge for 3 minutes at MAX speed and discard collection tubes. Place the spin column in a new microcentrifuge tube</li>
<li>Pipette 30 uL of Elution Buffer right above the membrane of the column without touching the membrane with the pipette tip. Let the column sit for 2 minutes.</li>
<li>Centrifuge for 60 seconds at MAX speed</li>
<li>Nanodrop DNA for concentration and store in at -20℃</li>
</ol>
</div>
<a class="anchor" name="PCRCleanup"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">PCR Cleanup</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>1.7ml Microcentrifuge Tubes</li>
<li>Spin Columns</li>
<li>Spin Tubes</li>
<li>PX Buffer</li>
<li>WN Buffer</li>
<li>WS Buffer</li>
<li>Centrifuge</li>
<li>Vacuum Manifold</li>
<li>55℃ Bead Bath</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 10-100ul of PCR product into 1.7ml microcentrifuge tube</li>
<li>Add 500ul of PX buffer to microcentrifuge tube</li>
<li>Transfer contents to spin columns that are place on the vacuum manifold. Turn on manifold</li>
<li>Pipette 500 uL of WN buffer into each column</li>
<li>Pipette 500 uL of WS buffer into each column</li>
<li>Turn of vacuum manifold and place spin columns in collection tubes</li>
<li>Centrifuge for 3 minutes at MAX speed and discard collection tubes. Place the spin column in a new microcentrifuge tube</li>
<li>Pipette 30 uL of Elution Buffer right above the membrane of the column without touching the membrane with the pipette tip. Let the column sit for 2 minutes.</li>
<li>Centrifuge for 60 seconds at MAX speed</li>
<li>Nanodrop DNA for concentration and store in at -20℃</li>
</ol>
</div>
<a class="anchor" name="Digestion"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Digestion</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>Restriction Enzymes (keep on ice or ice block)</li>
<li>Restriction Enzyme Buffer</li>
<li>DNA</li>
<li>Deionized water</li>
<li>PCR Tubes</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 4ug of plasmid DNA or 2ug of linear DNA to PCR tube</li>
<li>Total amount of enzymes added should be 4ul</li>
<li>Add 5ul of compatible buffer for restriction enzymes</li>
<li>Add deionized water to a total volume of 50ul</li>
<li>Place in thermocycler</li>
<li>If adding CIP- add 1ul of CIP to the sample in the final 20 minutes while the sample remains in the thermocycler</li>
</ol>
</div>
<a class="anchor" name="Ligation"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Ligation</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>T4 DNA Ligase (keep on ice or ice block)</li>
<li>T4 DNA Ligase Buffer</li>
<li>DNA</li>
<li>Deionized Water</li>
<li>PCR Tubes</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add equimolar concentrations of DNA fragments to PCR tube</li>
<li>Add equimolar concentrations of DNA fragments to PCR tube</li>
<li>Add 1ul of T4 DNA ligase</li>
<li>Add deionized water to a final volume of 20ul</li>
<li>Leave on bench for 20 minutes</li>
<li>Heat inactivate by placing sample in 70℃ bead bath for 1 minute</li>
</ol>
</div>
<a class="anchor" name="Transformation"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Transformation</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>KCM</li>
<li>Top 10 Competent Cells</li>
<li>Deionized Water</li>
<li>PCR Tubes</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 10ul of KCM to sample</li>
<li>Add deionized water to 50ul</li>
<li>Transfer contents of sample to Top Ten Cells</li>
<li>Place in thermocycler when the block reaches 4℃</li>
<li>Once finished in thermocycler spread the entire sample onto plates</li>
</ol>
</div>
<a class="anchor" name="LiquidCultures"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Liquid Cultures</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>12ml Culture Tubes</li>
<li>Liquid Media</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 4ml of liquid media to culture tube</li>
<li>Pick colony and eject tip into culture tube</li>
<li>Incubate in a 37°C shaking incubator for 16-18 hours</li>
</ol>
</div>
<a class="anchor" name="TestCuts"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Test Cuts</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>Restriction Enzymes</li>
<li>Restriction Enzyme Buffer</li>
<li>DNA</li>
<li>Deionized Water</li>
<li>Microcentrifuge Tubes</li>
<li>PCR Tubes</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Make a master mix with the following volumes in a microcentrifuge tube: 1ul of Buffer, 0.25ul of each Restriction Enzyme, 6.5ul of Deionized Water</li>
<li>Add 2ul of DNA to 8ul of Master Mix in a PCR tube</li>
<li>Incubate reaction at 37°C for at least 15-30 minutes</li>
<li>Run on agarose gel</li>
</ol>
</div>
<a class="anchor" name="Gibson"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Gibson</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>DNA insert</li>
<li>DNA backbone</li>
<li>Deionized Water</li>
<li>Gibson Master Mix</li>
<li>PCR tubes</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add equimolar concentrations of DNA insert and DNA backbone to PCR tube</li>
<li>Add deionized water to a total volume of 10ul</li>
<li>Add 10ul of Gibson Master Mix</li>
<li>Place in thermocycler</li>
</ol>
</div>
<a class="anchor" name="Recombination"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Recombination Reaction</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>250ng of DNA</li>
<li>10X of Recombinase Buffer</li>
<li>Recombinase</li>
<li>Deionized Water</li>
<li>PCR Tubes</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 250ng of DNA</li>
<li>Add 5ul of 10X Recombinase Buffer</li>
<li>Add 1ul of Recombinase</li>
<li>Add deionized water to total volume of 50ul</li>
<li>Incubate reaction at 37°C for 30 minutes</li>
<li> Hit Shock at 70°C for 10 minutes</li>
</ol>
</div>
<a class="anchor" name="CellFree"></a>
<h3 class="inline-heading-type"> </h3>
<h3 class="inline-heading-type">Cell-Free Reaction</h3>
<div>
<p class="body-type">
Materials
</p>
<ul class="body-type">
<li>Cell Free Crude Cell Extract Master Mix</li>
<li>Autoclaved Water</li>
<li>DNA</li>
<li>384 Well Plates</li>
<li>PCR Tubes</li>
<li>Plate Reader </li>
<li>Ice</li>
</ul>
<p class="body-type">
Procedure
</p>
<ol class="body-type" type="1">
<li>Add 4nM of DNA to each PCR tube</li>
<li>Add 16.5ul of Cell Free Crude Cell Extract Master Mix to PCR tube</li>
<li>Add autoclaved water to total volume of 20ul</li>
<li> Centrifuge PCR tubes</li>
<li> Transfer reactions from PCR tubes to a 384 Well Plates</li>
<li>Take initial fluorescence reading using a plate reader with wavelengths at 485nm and 525nm</li>
<li>Incubate plate at 30°C</li>
<li> Measure fluorescence every hour, we found that fluorescence levels out at 6 hours</li>
</ol>
</div>
<a class="anchor" name="MakingCF"></a>
</div>
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