Difference between revisions of "Team:William and Mary/Protocols:Gibson"

Line 81: Line 81:
 
                             <a href="https://2015.igem.org/Team:William_and_Mary/Measurement">  
 
                             <a href="https://2015.igem.org/Team:William_and_Mary/Measurement">  
 
                             Measurement
 
                             Measurement
 +
                            </a>
 +
                        </li>
 +
                        <li>
 +
                            <a href="https://2015.igem.org/Team:William_and_Mary/Modeling">
 +
                            Modeling
 
                             </a>
 
                             </a>
 
                         </li>
 
                         </li>
Line 133: Line 138:
 
          
 
          
 
             <!--************************    replace this line with the code generated with the rubik-builder.html              *******************************-->
 
             <!--************************    replace this line with the code generated with the rubik-builder.html              *******************************-->
−
  <div class="section section-we-are-1">
+
  <div class="section section-we-are-1" style="padding-bottom: 0px; padding-top: 0px;">
−
     <div class="text-area">
+
     <div class="text-area" style="padding-top: 50px; padding-bottom: 35px;">
−
         <div class="container">
+
         <div class="container" height= "200">
 
             <div class="row">
 
             <div class="row">
 
                 <div class="title">
 
                 <div class="title">
−
                     <p class="h2WM">Gibson Assembly</p>
+
                     <p class="h2WM">Measurement</p>
−
                    <p class="large">All of our constructs were assembled with the Gibson Assembly method. Many of our primers were designed with the help of NEB’s Gibson Primer builder, NEBuilder.NEB.com. <br>
+
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>  
  
−
Our PCRs to assemble were done with NEB’s HotStart Master Mix. The NEB recommended protocol can be found here. <br>
 
  
−
We often used the following protocol for our PCRs: <br>    2.5 µl Primer 1<br>
+
<object data="https://static.igem.org/mediawiki/2015/b/b8/WMnoise.pdf" type="application/pdf" width="100%" height="700px">
−
    2.5 µl Primer 2<br>
+
  <p>It appears you don't have a PDF plugin for this browser.
−
    1 µl of 1:100 dilution of miniprep template DNA<br>
+
   No biggie... you can <a href="https://static.igem.org/mediawiki/2015/4/49/Gibson.pdf">click here to
−
    6.5 µl NFW<br>
+
  download the PDF file.</a></p>
−
    12.5 µl HotStart Master Mix<br><br>
+
</object>
 +
</div>
 +
     
  
−
Many times, we altered the amount of primer, water, and template DNA as necessary to get PCRs and assemblies to work.<br> <br>
 
  
−
We would then run the PCR products on a gel to confirm that they were the correct size, and if so, would perform a DpnI digestion to remove residual template DNA: <br>
+
<footer class="footer footer-color-black" data-color="black">
−
24 µl PCR product (the entire volume after running the gel) <br>
+
                            <div class="container">
−
2.7 µl CutSmart Buffer <br>
+
                                <nav class="pull-left navbar-burger">
−
0.5 µl DpnI <br> <br>
+
                                    <ul>
−
 
+
                                        <li>
−
After the DpnI, we would either PCR-Purify or perform a Gel Extraction, depending on if there were extra multiple bands on the gel that we wanted to remove. We followed these protocols as described by QIAGEN, with the one addition of heating Buffer EB to 60°C before eluting to increase yields. <br>
+
                                            <a href="#">
−
 
+
                                                Home
−
From here, we proceeded to the Gibson reaction. We used NEB’s HiFi Assembly Master Mix for our assemblies. We reduced the reaction volume by half, in order to conserve Master Mix. <br>
+
                                            </a>
−
 
+
                                        </li>
−
Finally, we performed a chemical transformation with NEB’s 5-alpha Competent E. coli with 2 µl Gibson product. <br>
+
                                        <li>
−
</p>
+
                                            <a href="#">
−
<p> link to download software here.</p>
+
                                                Contact
−
 
+
                                            </a>
−
 
+
                                        </li>
−
                </div>
+
                                        <li>
−
            </div>
+
                                            <a href="#">
−
        </div>
+
                                              Blog
−
    </div>
+
                                            </a>
−
</div>                  
+
                                        </li>
 +
                                    </ul>
 +
                                </nav>
 +
                                <div class="social-area pull-right">               
 +
                                    <a class="btn btn-social btn-facebook btn-simple">
 +
                                        <i class="fa fa-facebook-square"></i>
 +
                                    </a>
 +
                                    <a class="btn btn-social btn-twitter btn-simple">
 +
                                        <i class="fa fa-twitter"></i>
 +
                                    </a>
 +
                                </div>
 +
                                <div class="copyright">
 +
                                    © 2015 William & Mary iGEM
 +
                                </div>
 +
                            </div>
 +
                        </footer>      
 
                  
 
                  
 
     </body>
 
     </body>

Revision as of 22:34, 18 September 2015

NOISE - W&M iGEM

Measurement

It appears you don't have a PDF plugin for this browser. No biggie... you can click here to download the PDF file.