Team:Cooper Union/Protocols

From 2014.igem.org

(Difference between revisions)
Line 3: Line 3:
<title>Cooper Union 2014 iGEM</title>
<title>Cooper Union 2014 iGEM</title>
<body>
<body>
-
 
+
<a name="top"></a>
<div class="parent-container">
<div class="parent-container">
Line 110: Line 110:
<hr>
<hr>
<br><br>
<br><br>
-
<a name="digest"><h2>Restriction Enzyme Digest</h2>
+
<a name="digest"></a><h2>Restriction Enzyme Digest</h2>
<br><br>
<br><br>
 +
<dl><dt>Materials</dt>
 +
<dd>10x Buffer (Compatible to the enzyme)</dd>
 +
<dd>Enzyme</dd>
 +
<dd>BSA - diluted to 10x</dd>
 +
<dd>ddH2O</dd>
 +
<dd>DNA - up to 1&mu;g per reaction.</dd>
 +
</dl><br>
-
 
+
<b>Procedure</b>
 +
<em>Verify all times and temperatures for specific enzyme prior to use. This is general protocol.</em>
 +
<ol>
 +
<li>Prewarm 2 water baths to 37&deg;C and 70&deg;C</li>
 +
<li>Thaw buffers, diluted BSA. Keep all reagents on ice</li>
 +
<li>Set up reactions based on table below.     
 +
<table>
 +
<tr><td>Reagent</td><td>Volume</td><td>Example</td></tr>
 +
<tr><td>Reaction Buffer</td><td>1/10 total volume</td><td align="right">2&mu;L</td></tr>
 +
<tr><td>Diluted BSA (10x)</td><td>1/10 total volume</td><td align="right">2&mu;L</td></tr>
 +
<tr><td>DNA</td><td>calculate for target ng</td><td align="right">3.7&mu;L</td></tr>
 +
<tr><td>ddH2O</td><td>volume up to (total volume - 1&mu;L)</td><td align="right">11.3&mu;L</td></tr>
 +
<tr><td>Enzyme</td><td>generally 1&mu;L</td><td align="right">1&mu;L</td></tr>
 +
<tr><td>Total Volume</td><td></td><td align="right">20uL</td></tr></table></li>
 +
<li>Incubate 60min 37°C.</li>
 +
<li>Heat inactive 70°C for 15 min.</li>
 +
<li>Store in -20°C or use immediately.</li>
 +
</ol>
 +
<br><br>
 +
<a href="#top">Top of Page</a>
 +
<br>
<hr>
<hr>

Revision as of 23:26, 14 October 2014

Cooper Union 2014 iGEM




Common Laboratory Protocols









Bacterial Transformation






Colony PCR






Gel Extraction






Miniprep






PCR Purification






PCR Reaction






Restriction Enzyme Digest



Materials
10x Buffer (Compatible to the enzyme)
Enzyme
BSA - diluted to 10x
ddH2O
DNA - up to 1μg per reaction.

Procedure Verify all times and temperatures for specific enzyme prior to use. This is general protocol.
  1. Prewarm 2 water baths to 37°C and 70°C
  2. Thaw buffers, diluted BSA. Keep all reagents on ice
  3. Set up reactions based on table below.
    ReagentVolumeExample
    Reaction Buffer1/10 total volume2μL
    Diluted BSA (10x)1/10 total volume2μL
    DNAcalculate for target ng3.7μL
    ddH2Ovolume up to (total volume - 1μL)11.3μL
    Enzymegenerally 1μL1μL
    Total Volume20uL
  4. Incubate 60min 37°C.
  5. Heat inactive 70°C for 15 min.
  6. Store in -20°C or use immediately.


Top of Page



TdT Heat Inactivation






TdT Nucleotide Addition






Yeast Gene Knockout






Yeast Mating






Yeast Sporulation/Zymolyase Treatment