Difference between revisions of "Part:BBa K1429002"
 
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<partinfo>BBa_K1429002 short</partinfo>
 
<partinfo>BBa_K1429002 short</partinfo>
  
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IP free cyan fluorescent protein.  We have a ribosome binding site (RBS).
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IP free synthetic non-Aequorea cyan fluorescent protein with ribosome binding site.  
  
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<h4>Background</h4>
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===Usage and Biology===
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<div class='entry-results'><p>We want to take Tuesday&#39;s PCR products and put them into the pSB1C3 backbone.&nbsp;</p>
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Can be used as a reporter. Production of CFP in E. coli under the control of a T7 promoter (part subcloned into pUC19) shown here as "CFP1":
  
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<p><strong>Digest PCRs:</strong></p>
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https://static.igem.org/mediawiki/2014/b/bc/UV_light.jpg
  
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<p>10 ul PCR product</p>
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https://static.igem.org/mediawiki/2014/d/d7/20141015_174513.jpg
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<p>2 ul cutsmart buffer (10x stock)</p>
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<p>1 ul PstI</p>
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<p><u>1 ul EcoRI</u></p>
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<p>20 ul total --&gt; incubate for 30 min at 37C</p>
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<p>&nbsp;</p>
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<p><strong>PCR purify digest product (only 14 ul - save 6 ul):</strong></p>
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<p>Follow kit protocol. Elute in elution buffer.</p>
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<p>Worried that the washed columns won&#39;t bind DNA, we are going to use some of the set-aside (unpurified) digest product for a backup ligation. We&#39;ll run a gel of our purification, but we are going to set up a ligation beforehand, so we won&#39;t have even rough estimates of DNA concentrations.</p>
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<p>Set up ligations:</p>
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<table style="width: 500px;">
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<tbody>
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<tr>
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<td><strong>Component</strong></td>
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<td><strong>Using purified digest product</strong></td>
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<td><strong>Using unpurified digest product</strong></td>
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<td><strong>BB alone</strong></td>
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</tr>
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<tr>
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<td>dH2O</td>
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<td>x</td>
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<td>11</td>
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<td>14</td>
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</tr>
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<tr>
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<td>Insert (RFP or GFP)</td>
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<td>14</td>
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<td>3</td>
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<td>x</td>
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</tr>
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<tr>
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<td>1:10 BB</td>
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<td>3</td>
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<td>3</td>
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<td>3</td>
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</tr>
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<tr>
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<td>T4 buffer (10</td>
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<td>1</td>
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<td>1</td>
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<td>1</td>
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</tr>
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<tr>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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</tr>
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</tbody>
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</table>
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<p>The above were incubated 30 min at RT then stored at -20C.</p>
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<p>&nbsp;</p>
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</div>
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<br>
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</div>
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<div class='entry'>
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<h4>Results</h4>
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<div class='entry-results'><p>We ran a 1% gel of the digest before and after purification. We had a decent yield, maybe 40% of our initial digest product in the purified lanes.&nbsp;</p>
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</div>
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<br>
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</div>
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<div class='entry'>
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<h4>Conclusions</h4>
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<div class='entry-results'><p>Next step: Transform the ligated plasmids into E. coli!</p>
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</div>
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<br>
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</div>
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<!-- -->
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The excitation and emission spectra match the specifications on the DNA2.0 website. See 'experience' tab above for spectra.
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1429002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1429002 SequenceAndFeatures</partinfo>

Latest revision as of 00:28, 22 October 2014

Cyan Fluorescent Protein (CFP) "Cindy Lou" coding region, intellectual property-free

IP free synthetic non-Aequorea cyan fluorescent protein with ribosome binding site.

Usage and Biology

Can be used as a reporter. Production of CFP in E. coli under the control of a T7 promoter (part subcloned into pUC19) shown here as "CFP1":

UV_light.jpg

20141015_174513.jpg

The excitation and emission spectra match the specifications on the DNA2.0 website. See 'experience' tab above for spectra. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 74
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2