IP free cyan fluorescent protein. We have a ribosome binding site (RBS).
IP free cyan fluorescent protein. We have a ribosome binding site (RBS).
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<h4>Background</h4>
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<div class='entry-results'><p>We want to take Tuesday's PCR products and put them into the pSB1C3 backbone. </p>
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<p><strong>Digest PCRs:</strong></p>
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<p>10 ul PCR product</p>
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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 --> incubate for 30 min at 37C</p>
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<p> </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't bind DNA, we are going to use some of the set-aside (unpurified) digest product for a backup ligation. We'll run a gel of our purification, but we are going to set up a ligation beforehand, so we won't have even rough estimates of DNA concentrations.</p>
<p>The above were incubated 30 min at RT then stored at -20C.</p>
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<p> </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. </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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Revision as of 00:54, 18 October 2014
Cyan Fluorescent Protein (CFP) "Cindy Lou" coding region, intellectual property-free
IP free cyan fluorescent protein. We have a ribosome binding site (RBS).
