Difference between revisions of "Team:UMaryland/protocols"

 
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margin:auto;
 
margin:auto;
 
font-family: "Palatino Linotype", "Book Antiqua", Palatino, serif;
 
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 +
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#cover {
 
#cover {
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margin:auto;
 
margin:auto;
 
padding:0px;
 
padding:0px;
−
background-image: url("https://static.igem.org/mediawiki/2015/9/97/Resultsq.png");
+
background-image: url("https://static.igem.org/mediawiki/2015/e/ed/Notebookumd.jpeg");
 
background-size: 100% ;
 
background-size: 100% ;
 
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background-repeat: no-repeat;
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font-size:xx-large;
 
font-size:xx-large;
 
top:-200px;
 
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 +
z-index:3;
 
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−
 
  
 
#bar{
 
#bar{
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height:200px;
 
height:200px;
 
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−
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+
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−
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+
 
 +
ul.a {list-style-type: circle;font-size:18px;color:black}
 
</style>
 
</style>
  
Line 124: Line 223:
 
<div id='bar'>
 
<div id='bar'>
 
<p style="font-size:64px"><b>Protocols</b>
 
<p style="font-size:64px"><b>Protocols</b>
 +
</div>
 
</div>
 
</div>
  
  
−
</div>
+
<div id='yolo'>
 +
<div id='sidemenu'>
 +
<ul>
 +
 
 +
<li><a href="#Miniprep" class="smoothScroll"><span>Miniprep</span></a></li>
 +
 
 +
<li><a href="#Ligation" class="smoothScroll"><span>Ligation</span></a></li>
 +
 
 +
<li><a href="#Transformation" class="smoothScroll"><span>Transformation</span></a></li>
 +
 
 +
<li><a href="#3Assembly" class="smoothScroll"><span>3A Assembly</span></a></li>
 +
 
 +
<li><a href="#REDigest" class="smoothScroll"><span>RE Digest</span></a></li>
 +
 
 +
<li><a href="#Gibsonassembly" class="smoothScroll"><span>Gibson Assembly</span></a></li>
 +
 
 +
<li><a href="#PCR" class="smoothScroll"><span>PCR</span></a></li>
 +
 
 +
<li><a href="#Gelextract" class="smoothScroll"><span>Gel Extract</span></a></li>
 +
<li><a href="#FPLC" class="smoothScroll"><span>FPLC</span></a></li>
 +
</ul>
 
</div>
 
</div>
  
  
 
<div id='contentbox'>
 
<div id='contentbox'>
 +
<a name="Miniprep">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Miniprep</b></a>
 
<b>Miniprep</b></a>
Line 168: Line 289:
  
 
<div id='contentbox'>
 
<div id='contentbox'>
 +
<a name="Ligation">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Ligation</b></a>
 
<b>Ligation</b></a>
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<div id='contentbox'>
 
<div id='contentbox'>
 +
<a name="Transformation">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Transformation</b></a>
 
<b>Transformation</b></a>
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   <li>- Add DNA to cells</li>
 
   <li>- Add DNA to cells</li>
 
   <li>- Incubate on ice for 30 minutes</li>
 
   <li>- Incubate on ice for 30 minutes</li>
−
   <li>- Heat shock at 42° fro 30 seconds</li>
+
   <li>- Heat shock at 42°C fro 30 seconds</li>
 
   <li>- Add 1 mL SOC media to the cells</li>
 
   <li>- Add 1 mL SOC media to the cells</li>
−
   <li>- Incubate for 60 minutes at 37°</li>
+
   <li>- Incubate for 60 minutes at 37°C</li>
 
   <li>- Plate 200 µL </li>
 
   <li>- Plate 200 µL </li>
−
   <li>- Incubate at 37°</li>
+
   <li>- Incubate at 37°C</li>
 
    
 
    
 
</ul>
 
</ul>
 
<br>
 
<br>
 
<br>
 
<br>
 +
</div>
 +
<div id='contentbox'>
 +
<a name="3Assembly">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>3A Assembly</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 5 µL CutSmart</li>
 +
  <li>- 0.5 µL BSA </li>
 +
  <li>- 0.5 µL upstream digest (DH5alpha)</li>
 +
  <li>- 0.5 µL downstream digest </li>
 +
  <li>- Add DDH2O to 20 µL</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a PCR tube</li>
 +
  <li>- Place in the thermocycler</li>
 +
  <li>- 37°C for 30 minutes</li>
 +
  <li>- 80°C for 20 minutes</li> 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<a name="REDigest">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>RE Digest</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 1 µg DNA</li>
 +
  <li>- 5 µL 10X NEBuffer </li>
 +
  <li>- Add DDH2O to 20 µL</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a PCR tube</li>
 +
  <li>- Incubate for 1 hour</li> 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<a name="Gibsonassembly">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Gibson Assembly</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 0.02-0.5 pmols of DNA</li>
 +
  <li>- 10 µL Gibson Assembly MasterMix (2X) </li>
 +
  <li>- Add DDH2O to 20 µL</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a PCR tube</li>
 +
  <li>- Place tube in thermocycler for 15 minutes at 50°C</li> 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<a name="PCR">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>PCR</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 4 µL Phusion HF/GC Buffer</li>
 +
  <li>- 0.4 µL 10 mM dNTPs</li>
 +
  <li>- 1 µL 10 µM forward primer</li>
 +
  <li>- 1 µL 10 µM forward primer</li>
 +
  <li>- 0.2 µL Phusion DNA polymerase</li>
 +
  <li>- (OPTIONAL) 0.6 µL DMSO</li>
 +
  <li>- <250 ng Template DNA </li>
 +
  <li>- Add DDH2O to 20 µL </li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a PCR tube</li>
 +
  <li>- Place tube in thermocycler for:</li> 
 +
  <li>- Initial Denaturation: 98° C for 30 seconds</li> 
 +
  <li>- 25-35 cycles: 98°C for 5-10 seconds, 45-72°C for 10-30 seconds and 72°C for 15-30 seconds per kb</li> 
 +
  <li>- Final Extension: 72°C</li> 
 +
  <li>- Hold 4-10°C</li> 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
<div id='contentbox'>
 +
<a name="Gelextract">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Gel Extract</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Cut out gel portions with scalpel</li>
 +
  <li>- Weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel</li>
 +
  <li>- Incubate at 50 for 10 mins until gels slice dissolves. Mix by vortexing tube every 2-3 mins during incubation to help the gel dissolve
 +
</li>
 +
  <li>- After the gel dissolves completely, check color of the mixture is yellow (it may be orange or violet)
 +
</li>
 +
  <li>- Add 10 µL of 3M sodium acetate (ph 5.0) to make solution more yellow (regardless of current solution color)</li>
 +
  <li>- The color of solution relates to ph indicator in Q3</li>
 +
  <li>- Add 1 gel volume of of isopropyl to the sample and mix. This increases yield of DNA fragments between 500 bp and 4KB</li>
 +
  <li>- Place a Q1Aquick spin column in a provided 2ml collection tube</li>
 +
  <li>- Apply sample to spin column, centrifuge for 1 min to bind dna</li>
 +
  <li>- Discard flow through</li>
 +
  <li>- Wash with .75 mL buffer PE in column, centrifuge 1 minute</li>
 +
  <li>- Place column in a clean 1.5 microcentrifuge tube</li>
 +
  <li>- 40 microliters DDH20 , let stand 1 minute, centrifuge 1 min</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
<div id='contentbox'>
 +
<a name="FPLC">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Fast Protein Liquid Chromatography</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Centrifuge cell lysate (balance with equal mass of water) for 60 minutes at 25000 rpm</li>
 +
  <li>- Wash FPLC column with equilibration column (X2) with  syringe</li>
 +
  <li>- Wash out FLPC system with equilibration buffer</li>
 +
  <li>- Wash affinity column with equilibration column with  syringe</li>
 +
  <li>- Wash affinity  column with cobalt acetate with syringe</li>
 +
  <li>- Wash off excess cobalt with syringe</li>
 +
  <li>- Connect affinity column to FPLC system</li>
 +
  <li>- Inject supernatant</li>
 +
  <li>- Wash FPLC column with equilibration column (X2) with syringe</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 
</div>
 
</div>
 
</div>
 
</div>
  
 
</html>
 
</html>

Latest revision as of 02:32, 19 September 2015