Team:USTC CHINA/Notebook/Protocols/Colony PCR

From 2013.igem.org

(Difference between revisions)
 
(2 intermediate revisions not shown)
Line 7: Line 7:
<div class="bar" align="center">
<div class="bar" align="center">
     <div class="container" align="left">
     <div class="container" align="left">
-
        <div id="tlogo"><img src="https://static.igem.org/mediawiki/2013/f/f8/2013ustc-china_T-VACCINE.png" width="100%" height="123" />
 
-
        </div>
 
<div id="igemlogo"><a href="https://2013.igem.org/Main_Page" target="_blank"><img src="https://static.igem.org/mediawiki/2013/2/26/2013ustcigem_IGEM_basic_Logo.png" alt="igem home page" width="50" height="40" /></a></div>
<div id="igemlogo"><a href="https://2013.igem.org/Main_Page" target="_blank"><img src="https://static.igem.org/mediawiki/2013/2/26/2013ustcigem_IGEM_basic_Logo.png" alt="igem home page" width="50" height="40" /></a></div>
 +
<ul id="nav">
<ul id="nav">
-
          
+
         <li><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a></li>
-
<ul id="nav">
+
        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Overview">Project</a>
-
                <li><a href="https://2013.igem.org/Team:USTC_CHINA" target="_blank">Home</a></li>
+
            <ul class="subs">
-
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project" target="_blank">Project</a>
+
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Overview">Overview</a></li>
-
                    <ul class="subs">
+
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project/ProjectDetails">Project Details</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Overview" target="_blank">Overview</a></li>
+
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Results">Results</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project/ProjectDetails" target="_blank">Project Details</a></li>
+
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Parts">Parts</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Project/Results" target="_blank">Results</a></li>
+
            </ul>
-
                    </ul>
+
        </li>
-
                </li>
+
        <li class="active"><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">Notebook</a>
-
                <li class="active"><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook" target="_blank">Notebook</a>
+
              <ul class="subs">
-
                    <ul class="subs">
+
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Timeline">Timeline</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Timeline" target="_blank">Timeline</a></li>
+
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">Protocols</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols" target="_blank">Protocols</a></li>
+
              </ul>
-
                    </ul>
+
        </li>
-
                </li>
+
        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/">Modeling</a>
-
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/" target="_blank">Modeling</a>
+
              <ul class="subs">
-
                    <ul class="subs">
+
                  <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/KillSwitch">Kill Switch</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/ReporterSystem" target="_blank">Reporter System</a></li>
+
                  <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/B.SubtilisCulture">B.Subtilis Culture</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/B.SubtilisCulture" target="_blank">B.Subtilis Culture</a></li>
+
                  <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/DesignsofImmuneExperiments">Designs of Immune Experiments</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/MiceModeling" target="_blank">Mice Modeling</a></li>
+
              </ul>
-
                    </ul>
+
        </li>
-
              </li>
+
        <li><a href="https://2013.igem.org/Team:USTC_CHINA/HumanPractice">Human Practice</a>
-
              <li><a href="https://2013.igem.org/Team:USTC_CHINA/HumanPractice" target="_blank">Human Practice</a>
+
              <ul class="subs">
-
                    <ul class="subs">
+
                  <li><a href="https://2013.igem.org/Team:USTC_CHINA/HumanPractice/Communication" >Communication</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/HumanPractice/Communication" target="_blank">Communication</a></li>
+
                  <li><a href="https://2013.igem.org/Team:USTC_CHINA/HumanPractice/Activity">Activity</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/HumanPractice/Activity" target="_blank">Activity</a></li>
+
              </ul>
-
                    </ul>
+
        </li>
-
              </li>
+
        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Team">Team</a>
-
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Team" target="_blank">Team</a>
+
              <ul class="subs">
-
                <ul class="subs">
+
                  <li><a href="https://2013.igem.org/Team:USTC_CHINA/Team">Members</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Team/Members" target="_blank">Members</a></li>
+
                  <li><a href="https://igem.org/Team.cgi?year=2013&team_name=USTC_CHINA">Profile</a></li>
-
                        <li><a href="https://igem.org/Team.cgi?year=2013&team_name=USTC_CHINA" target="_blank">Profile</a></li>
+
                  <li><a href="https://2013.igem.org/Team:USTC_CHINA/Attributions">Attributions</a></li>
-
                        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Team/Attribute" target="_blank">Attribute</a></li>
+
                  <li><a href="https://igem.org/2013_Judging_Form?id=1074#iGEM_Medals">Achievements</a></li>
-
                  </ul>
+
              </ul>
-
              </li>
+
        </li>
-
                <li><a href="https://2013.igem.org/Team:USTC_CHINA/Safety">Safety</a></li>
+
        <li><a href="https://2013.igem.org/Team:USTC_CHINA/Safety">Safety</a></li>
-
            </ul>
+
    </ul>
 +
        <div id="tlogo"><img src="https://static.igem.org/mediawiki/2013/f/f8/2013ustc-china_T-VACCINE.png" width="100%" height="123" />
 +
        </div>
         </div>
         </div>
         </div>
         </div>
<div class="content" align="center">
<div class="content" align="center">
<div class="conbar1">
<div class="conbar1">
-
<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Colony PCR">protocols Colony PCR</a></div></div>
+
<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">Notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">Protocols</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Colony PCR">Colony PCR</a></div></div>
<div class="conbar2">
<div class="conbar2">
<div class="leftbar" align="left">
<div class="leftbar" align="left">
Line 100: Line 101:
   </tr>
   </tr>
</table>
</table>
 +
</br>
*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:</br>
*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:</br>
 +
</br>
<table width="580" border="2">
<table width="580" border="2">
   <tr>
   <tr>
Line 180: Line 183:
<div class="rightbar">
<div class="rightbar">
-
<div class="port-sidebar-border"><h>notebook</h></div>
+
<div class="port-sidebar-border"><h>Notebook</h></div>
<div class="clear"></div>
<div class="clear"></div>
-
<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Timeline">timeline</a></div>
+
<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Timeline">Timeline</a></div>
-
<div id="t1"><a class="active" href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a></div></div>
+
<div id="t1"><a class="active" href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">Protocols</a></div></div>
</div>
</div>
</div>
</div>
</body>
</body>
</html>
</html>

Latest revision as of 08:56, 27 September 2013

Colony PCR

Performed with Thermo Scientific Taq DNA Polymerase (recombinant), 5U/μl

Volume (μl)
10×Taq Buffer 5
dNTP Mix, 2 mM each 5 (0.2 mM of each)
Forward primer 0.1-1.0 μM
Reverse primer 0.1-1.0 μM
25 mM MgCl2* 1-4 mM
Template DNA Picked from the colony after transformation
Taq DNA Polymerase 0.25 U
ddH2O To 10
Total 10

*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:

Final concentration of MgCl2(mM) 1 1.5 2 2.5 3 4
Volume of 25 mM MgCl2 to be added for 10 μl reaction(μl) 0.4 0.6 0.8 1.0 1.2 1.6

3. Gently vortex the samples and spin down.
4. Perform PCR using recommended thermal cycling conditions:

Step Temperature, °C Time Number of cycles
Initial denaturation 95 10 min 1
Denaturation 95 30s 25~40
Annealing Tm-5 30s 25~40
Extension 72 1 min/kb 25~40
Final Extension 72 5-15 min 1

Notebook

Retrieved from "http://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Colony_PCR"