Difference between revisions of "Team:Cadets2Vets/Experiments"

Line 11: Line 11:
 
{{Cadets2Vets}}
 
{{Cadets2Vets}}
 
<html>
 
<html>
 +
<body class="header-page  wsite-theme-light  wsite-page-about"><div class="wrapper">
 +
  <div class="header-wrap">
 +
    <div id="topBar" class="topbar">
 +
      <label class="hamburger"><span></span></label>
 +
      <div class="logo"><span class="wsite-logo">
 +
 +
 +
<span class="wsite-title-placeholder">&nbsp;</span><span style="display:none">
 +
<span style="display:none">Acquire</span>
 +
</span>
 +
 +
 +
</span></div>
 +
         
 +
         
 +
    </div><!-- end .container -->
 +
 +
  <div class="banner-wrap">
 +
<div class="wsite-elements wsite-not-footer wsite-header-elements">
 +
<div class="wsite-section-wrap">
 +
<div  class="wsite-section wsite-header-section wsite-section-bg-image" style="height: 289.99998474121093px;vertical-align: middle;background-image: url(&quot;/uploads/1/0/7/7/107726947/background-images/2072709013.jpg&quot;) ;background-repeat: no-repeat ;background-position: 50.00% 86.68% ;background-size: 100% ;background-color: transparent ;background-size: cover;" >
 +
<div class="wsite-section-content">
 +
 +
<div class="container">
 +
<div class="banner">
 +
<div class="wsite-section-elements">
 +
<h2 class="wsite-content-title">Experiments</h2>
 +
</div>
 +
</div>
 +
</div>
 +
 +
</div>
 +
<div class=""></div>
 +
</div>
 +
</div>
 +
 +
</div>
 +
 +
 +
</div>
 +
  </div><!-- end .header-wrap -->
 +
 +
<div class="w-navlist nav-wrap">
 +
  <div class="container">
 +
  <div class="nav desktop-nav"><ul class="wsite-menu-default">
 +
<li id="pg512017008766558506" class="wsite-menu-item-wrap">
 +
<a
 +
href="/"
 +
class="wsite-menu-item"
 +
>
 +
Home
 +
</a>
 +
 +
</li>
 +
<li id="pg222259470211333982" class="wsite-menu-item-wrap">
 +
<a
 +
href="/design.html"
 +
class="wsite-menu-item"
 +
>
 +
design
 +
</a>
 +
 +
</li>
 +
<li id="active" class="wsite-menu-item-wrap">
 +
<a
 +
href="/about.html"
 +
class="wsite-menu-item"
 +
>
 +
About
 +
</a>
 +
 +
</li>
 +
<li id="pg736091085660923040" class="wsite-menu-item-wrap">
 +
<a
 +
href="/news.html"
 +
class="wsite-menu-item"
 +
>
 +
News
 +
</a>
 +
 +
</li>
 +
<li id="pg581528279135988304" class="wsite-menu-item-wrap">
 +
<a
 +
href="/contact.html"
 +
class="wsite-menu-item"
 +
>
 +
Contact
 +
</a>
 +
 +
</li>
 +
</ul>
 +
</div>
 +
  </div>
 +
</div>
 +
 +
<div class="main-wrap">
 +
<div id="wsite-content" class="wsite-elements wsite-not-footer">
 +
<div class="wsite-section-wrap">
 +
<div class="wsite-section wsite-body-section wsite-background-5 wsite-custom-background" style="height: auto;" >
 +
<div class="wsite-section-content">
 +
    <div class="container">
 +
<div class="wsite-section-elements">
 +
<h2 class="wsite-content-title" style="text-align:center;"><font size="2">Protocols </font><br /></h2>
 +
 +
<div class="paragraph" style="text-align:center;">Our project required us to utilize different "protocols" or experimental procedures in order to successfully run our circuit. <br /></div>
 +
</div>
 +
</div>
 +
</div>
 +
 +
</div>
 +
</div>
 +
<div class="wsite-section-wrap">
 +
<div class="wsite-section wsite-body-section wsite-section-bg-color wsite-background-6 wsite-custom-background" style="height: auto;background-color: #ffffff;background-image: none;is_customized: 1;" >
 +
<div class="wsite-section-content">
 +
    <div class="container">
 +
<div class="wsite-section-elements">
 +
<div><div class="wsite-multicol"><div class="wsite-multicol-table-wrap" style="margin:0 -15px;">
 +
<table class="wsite-multicol-table">
 +
<tbody class="wsite-multicol-tbody">
 +
<tr class="wsite-multicol-tr">
 +
<td class="wsite-multicol-col" style="width:49.999999999999%; padding:0 15px;">
 +
 +
 +
 +
<h2 class="wsite-content-title" style="text-align:center;"><font size="4"><font size="5"><font color="#2a2a2a" size="3"><font size="4"><font size="5">Ticketed Cell Free Expression System</font></font></font></font></font><br /></h2>
 +
 +
<div><div class="wsite-image wsite-image-border-none " style="padding-top:0px;padding-bottom:10px;margin-left:0px;margin-right:0px;text-align:center">
 +
<a>
 +
<img src="/uploads/1/0/7/7/107726947/published/img-3798.jpg?1497553086" alt="Picture" style="width:auto;max-width:100%" />
 +
</a>
 +
<div style="display:block;font-size:90%"></div>
 +
</div></div>
 +
 +
<h2 class="wsite-content-title"><font size="2"><font size="3">Overview</font></font></h2>
 +
 +
<div class="paragraph"><font color="#000000">The idea of cell free expression has existed for more than 20 years. It is the notion of taking the elements required for both transcription and translation and utilizing them for an external protein expression system. This is extremely beneficial for expression of cytotoxic proteins in which high yields are difficult to produce in vivo. For our purpose within this assay, this concept is utilized in conjunction with a regulatory factor for detection. This is detection is predicated either by regulation of the RBS at the transcript level or by sequestering the binding sight of RNA polymerase. </font><br /></div>
 +
 +
<h2 class="wsite-content-title">work flow<br /></h2>
 +
 +
<div class="paragraph"><ol><li>Identify trigger sequence and target plasmid or probe</li><li>Add target</li><li>Incubation</li><li>&nbsp;Evaluation<br /></li></ol></div>
 +
 +
<h2 class="wsite-content-title">Reagents<br /></h2>
 +
 +
<div class="paragraph"><ul><li>Cell free expression kit<ul><li>Promega L5540</li><li>Promega L1020</li><li>Promega L1110</li><li>NEB E6800L</li><li>Life Tech K990100</li></ul></li><li>RNASE Inhibitor (RI)</li><li>Prepared tickets</li><li>Trigger Plasmid</li><li>Substrate if needed<br /></li></ul></div>
 +
 +
 +
 +
</td> <td class="wsite-multicol-col" style="width:49.999999999999%; padding:0 15px;">
 +
 +
 +
 +
<h2 class="wsite-content-title" style="text-align:center;"><font size="4"><font color="#2a2a2a" size="5">Qpcr<font color="#a9a9a9">- <font size="4"><br />Quantatative polymerase Chain reaction</font></font></font></font><br /></h2>
 +
 +
<div class="paragraph">qPCR utilizes the same basis as standard polymerase chain reaction, in which a target section of DNA can be identified and amplified via an enzymatic reaction (DNA Polymerase) and two ssDNA oligos (primers). Where qPCR differs is in the addition of either dye or probe based chemistry (this protocol will be primarily deal with the former). What this dye allows for is detection of newly formed DNA through the incorporation of a small fluorescent molecule into the new double stranded DNA (dsDNA). When the relative fluorescent units (RFU) being monitored rises above the background level (established by experimental controls) it is possible to ascertain a concentration of target gene. The relationship being the more concentrated the target the earlier in the thermocycler program signal will be generated.<br /></div>
 +
 +
<h2 class="wsite-content-title">Work Flow<br /></h2>
 +
 +
<div class="paragraph">1. Initial Prepartaion<br />2. Create master mix<br />3. Aliquot master mix<br />4. Add target DNA and standards to appropriate wells<br />5. Start thermocycler<br /></div>
 +
 +
<h2 class="wsite-content-title">reagents<br /></h2>
 +
 +
<div class="paragraph">1. ICE<br />2. Promega Hotstart Polyermase + Colorless Flexi buffer<br />3. 10 mM dNTP<br />4. 25 mM MgCl<br />5. Molecular Water<br />6. Forward and Reverse Primers in 10 uM<br />7. Evagreen dye 25x<br />8. Agarose<br />9. Sybr Safe<br /></div>
 +
 +
 +
 +
</td> </tr>
 +
</tbody>
 +
</table>
 +
</div></div></div>
 +
</div>
 +
</div>
 +
</div>
 +
 +
</div>
 +
</div>
 +
 +
</div>
 +
 +
  </div>
 +
 +
    </body>
 +
</html>
  
 
<div class="column full_size">
 
<div class="column full_size">

Revision as of 21:47, 15 June 2017






HOME
TEAM
PROJECT
Description
Design
Experiments
Notebook
InterLab
Contribution
Model
Results
Demonstrate
Improve
Attributions
PARTS
Parts
Basic Parts
Composite Parts
Part Collection
SAFETY
HUMAN PRACTICES
Silver HP
Integrated and Gold
Public Engagement
AWARDS
Applied Design
Entrepreneurship
Hardware
Measurement
Model
Plant
Software
JUDGING FORM

Cadets2Vets

Protocols

Our project required us to utilize different "protocols" or experimental procedures in order to successfully run our circuit.

Ticketed Cell Free Expression System

Overview

The idea of cell free expression has existed for more than 20 years. It is the notion of taking the elements required for both transcription and translation and utilizing them for an external protein expression system. This is extremely beneficial for expression of cytotoxic proteins in which high yields are difficult to produce in vivo. For our purpose within this assay, this concept is utilized in conjunction with a regulatory factor for detection. This is detection is predicated either by regulation of the RBS at the transcript level or by sequestering the binding sight of RNA polymerase.

work flow

  1. Identify trigger sequence and target plasmid or probe
  2. Add target
  3. Incubation
  4.  Evaluation

Reagents

  • Cell free expression kit
    • Promega L5540
    • Promega L1020
    • Promega L1110
    • NEB E6800L
    • Life Tech K990100
  • RNASE Inhibitor (RI)
  • Prepared tickets
  • Trigger Plasmid
  • Substrate if needed

Qpcr-
Quantatative polymerase Chain reaction

qPCR utilizes the same basis as standard polymerase chain reaction, in which a target section of DNA can be identified and amplified via an enzymatic reaction (DNA Polymerase) and two ssDNA oligos (primers). Where qPCR differs is in the addition of either dye or probe based chemistry (this protocol will be primarily deal with the former). What this dye allows for is detection of newly formed DNA through the incorporation of a small fluorescent molecule into the new double stranded DNA (dsDNA). When the relative fluorescent units (RFU) being monitored rises above the background level (established by experimental controls) it is possible to ascertain a concentration of target gene. The relationship being the more concentrated the target the earlier in the thermocycler program signal will be generated.

Work Flow

1. Initial Prepartaion
2. Create master mix
3. Aliquot master mix
4. Add target DNA and standards to appropriate wells
5. Start thermocycler

reagents

1. ICE
2. Promega Hotstart Polyermase + Colorless Flexi buffer
3. 10 mM dNTP
4. 25 mM MgCl
5. Molecular Water
6. Forward and Reverse Primers in 10 uM
7. Evagreen dye 25x
8. Agarose
9. Sybr Safe

Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

What should this page contain?
  • Protocols
  • Experiments
  • Documentation of the development of your project
Inspiration




</html>