Team:UC Davis/protocols/digestion.html

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                 <li>Sterilized milliQ water</li>
                 <li>Sterilized milliQ water</li>
                 <li>BSA</li>
                 <li>BSA</li>
-
                 <li>NEB buffers 1,2,3,and/or 4 </li>
+
                 <li>NEB buffers 1,2,3,and/or 4 (see extra notes)</li>
                 <li>Miniprepped sample or plasmid you wish to cut</li>
                 <li>Miniprepped sample or plasmid you wish to cut</li>
                 <li>NEB digestion enzymes (EcoRI, SpeI, PstI, NheI, and/or XbaI)</li>
                 <li>NEB digestion enzymes (EcoRI, SpeI, PstI, NheI, and/or XbaI)</li>
                 </ul><p>
                 </ul><p>
 +
<a name="extranotes"><h1>Extra Notes</h1></a>
 +
 +
Be sure to use the buffer that maximizes the compatibility and activity between the two enzymes. The numbers indicate the percentage of enzyme activity for each enzyme in each buffer.<p>
 +
 +
<table class="pikachu" class="marth" width="650px" padding="3px">
 +
<tr>
 +
<td>Enzyme</td>
 +
<td>Buffer 1</td>
 +
<td>Buffer 2</td>
 +
<td>Buffer 3</td>
 +
<td>Buffer 4</td>
 +
</tr>
 +
<tr>
 +
<td>EcoRI</td>
 +
<td>100</td>
 +
<td>100</td>
 +
<td>100</td>
 +
<td>100</td>
 +
</tr>
 +
<tr>
 +
<td>SpeI</td>
 +
<td>75</td>
 +
<td>100</td>
 +
<td>25</td>
 +
<td>100</td>
 +
</tr>
 +
<tr>
 +
<td>PstI</td>
 +
<td>75</td>
 +
<td>75</td>
 +
<td>100</td>
 +
<td>50</td>
 +
</tr>
 +
<tr>
 +
<td>NheI</td>
 +
<td>100</td>
 +
<td>100</td>
 +
<td>10</td>
 +
<td>100</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI</td>
 +
<td>0</td>
 +
<td>100</td>
 +
<td>75</td>
 +
<td>100</td>
 +
</tr>
 +
</table><p>
 +
<a name="procedure"><h1>Procedure</h1></a>
<a name="procedure"><h1>Procedure</h1></a>
 +
1. Add the following into a PCR tube <br />
 +
<ul>
 +
<li>22μL of milliQ water </li>
 +
<li>1μL BSA </li>
 +
<li>5μL buffer x (see extra notes) </li>
 +
<li>20μL template to be cut </li>
 +
<li>1μL digestion enzyme 1 </li>
 +
<li>1μL digestion enzyme 2
 +
</ul><p>
 +
2. Run in a thermocycler
<ul>
<ul>
-
<li>Add 22μL of milliQ water. </li>
+
<li>37°C for 3 hours </li>
 +
<li>80°C for 20 minutes </li>
 +
<li>Keep at 4°C if to be stored </li>
</ul>
</ul>
<p>
<p>
<a name="purpose"><h1>Purpose</h1></a>
<a name="purpose"><h1>Purpose</h1></a>
-
To cut DNA at the designated places.  
+
To cut DNA at the designated places; cut out the insert from the plasmid.
      
      
<a name="references"><h1>References</h1></a>
<a name="references"><h1>References</h1></a>
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<ul>
<ul>
<li><a href="#materials" class="help">Materials</a></li>
<li><a href="#materials" class="help">Materials</a></li>
 +
<li><a href="#extranotes" class="help">Extra Notes</a></li>
<li><a href="#procedure" class="help">Procedure</a></li>
<li><a href="#procedure" class="help">Procedure</a></li>
<li><a href="#purpose" class="help">Purpose</a></li>
<li><a href="#purpose" class="help">Purpose</a></li>

Latest revision as of 19:24, 9 September 2010

Digestion

Materials

You will need:

  • Sterilized milliQ water
  • BSA
  • NEB buffers 1,2,3,and/or 4 (see extra notes)
  • Miniprepped sample or plasmid you wish to cut
  • NEB digestion enzymes (EcoRI, SpeI, PstI, NheI, and/or XbaI)

Extra Notes

Be sure to use the buffer that maximizes the compatibility and activity between the two enzymes. The numbers indicate the percentage of enzyme activity for each enzyme in each buffer.

Enzyme Buffer 1 Buffer 2 Buffer 3 Buffer 4
EcoRI 100 100 100 100
SpeI 75 100 25 100
PstI 75 75 100 50
NheI 100 100 10 100
XbaI 0 100 75 100

Procedure

1. Add the following into a PCR tube
  • 22μL of milliQ water
  • 1μL BSA
  • 5μL buffer x (see extra notes)
  • 20μL template to be cut
  • 1μL digestion enzyme 1
  • 1μL digestion enzyme 2

2. Run in a thermocycler

  • 37°C for 3 hours
  • 80°C for 20 minutes
  • Keep at 4°C if to be stored

Purpose

To cut DNA at the designated places; cut out the insert from the plasmid.

References

We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)

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