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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). | + | IP free synthetic non-Aequorea cyan fluorescent protein with ribosome binding site. |
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| − | <h4>Background</h4>
| + | ===Usage and Biology=== |
| − | <div class='entry-results'><p>We want to take Tuesday's PCR products and put them into the pSB1C3 backbone. </p>
| + | 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>
| + | https://static.igem.org/mediawiki/2014/b/bc/UV_light.jpg |
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| − | <p>10 ul PCR product</p>
| + | https://static.igem.org/mediawiki/2014/d/d7/20141015_174513.jpg |
| − | | + | |
| − | <p>2 ul cutsmart buffer (10x stock)</p>
| + | |
| − | | + | |
| − | <p>1 ul PstI</p>
| + | |
| − | | + | |
| − | <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>
| + | |
| − | | + | |
| − | <p><strong>PCR purify digest product (only 14 ul - save 6 ul):</strong></p>
| + | |
| − | | + | |
| − | <p>Follow kit protocol. Elute in elution buffer.</p>
| + | |
| − | | + | |
| − | <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>
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| − | | + | |
| − | <p>Set up ligations:</p>
| + | |
| − | | + | |
| − | <table style="width: 500px;">
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td><strong>Component</strong></td>
| + | |
| − | <td><strong>Using purified digest product</strong></td>
| + | |
| − | <td><strong>Using unpurified digest product</strong></td>
| + | |
| − | <td><strong>BB alone</strong></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>dH2O</td>
| + | |
| − | <td>x</td>
| + | |
| − | <td>11</td>
| + | |
| − | <td>14</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Insert (RFP or GFP)</td>
| + | |
| − | <td>14</td>
| + | |
| − | <td>3</td>
| + | |
| − | <td>x</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>1:10 BB</td>
| + | |
| − | <td>3</td>
| + | |
| − | <td>3</td>
| + | |
| − | <td>3</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>T4 buffer (10</td>
| + | |
| − | <td>1</td>
| + | |
| − | <td>1</td>
| + | |
| − | <td>1</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td> </td>
| + | |
| − | <td> </td>
| + | |
| − | <td> </td>
| + | |
| − | <td> </td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | | + | |
| − | <p>The above were incubated 30 min at RT then stored at -20C.</p>
| + | |
| − | | + | |
| − | <p> </p>
| + | |
| − | </div>
| + | |
| − | <br>
| + | |
| − | </div>
| + | |
| − | <div class='entry'>
| + | |
| − | <h4>Results</h4>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | <br>
| + | |
| − | </div>
| + | |
| − | <div class='entry'>
| + | |
| − | <h4>Conclusions</h4>
| + | |
| − | <div class='entry-results'><p>Next step: Transform the ligated plasmids into E. coli!</p>
| + | |
| − | </div>
| + | |
| − | <br>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class='entry'>
| + | |
| − | <div class='entry-results'><p><strong>Background info:</strong></p>
| + | |
| − | | + | |
| − | <ul>
| + | |
| − | <li>The plasmid is about 2000 base pairs (bp). </li>
| + | |
| − | <li>Our PCRed GFP (gene of interest) is about 750 bp.</li>
| + | |
| − | <li>Assume that 1 uL of this PCR purified GFP = 50 ng (info from Ellen).</li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <p><strong>Calculations:</strong></p>
| + | |
| − | | + | |
| − | <p>We have 6 uL of our gene of interest in a solution totaling 20 uL.</p>
| + | |
| − | | + | |
| − | <p>So we have:</p>
| + | |
| − | | + | |
| − | <p><span class="math-tex">\(\displaystyle \frac{6\mu L }{1}PCR \; purified \; GFP * \frac{50 ng}{1 \mu L} * \frac{1}{20\mu L} = \frac{300 ng}{20 \mu L} PCR \; purified \: GFP = 15\frac{ng}{\mu L}PCR \; purified \; GFP\)</span></p>
| + | |
| − | | + | |
| − | <p> </p>
| + | |
| − | | + | |
| − | <p>The iGEM kit provides a linearized plasmid backbone in a solution with a concentration of 25 ng/uL. </p>
| + | |
| − | | + | |
| − | <p>In our digestion step, we used 4 uL of the linearized plasmid backbone and 4 uL of the enzyme master mix. </p>
| + | |
| − | | + | |
| − | <p>So, we have:</p>
| + | |
| − | | + | |
| − | <p><span class="math-tex">\(\displaystyle \frac{4\mu L }{1}plasmid * \frac{25 ng}{1 \mu L} * \frac{1}{(4+4)\mu L} = \frac{100 ng}{8 \mu L} plasmid= 12.5\frac{ng}{\mu L}plasmid\)</span></p>
| + | |
| − | | + | |
| − | <p>We need to have a GFP:plasmid ratio of at least 3:1 to make sure that we have enough pieces of the gene of interest to successfully connect to the plasmid backbone.</p>
| + | |
| − | | + | |
| − | <p>Now, we'll use the <a href="http://nebiocalculator.neb.com/#!/" target="_blank">NEBioCalculator</a> to get the number of moles for each (see the pictures).</p>
| + | |
| − | | + | |
| − | <p>So, we have:</p>
| + | |
| − | | + | |
| − | <p><span class="math-tex">\(\displaystyle \frac{32 . 36 \frac{fmol}{\mu L} \; GFP}{9 . 632 \frac{fmol}{\mu L} \; plasmid} = 3 . 36 \; GFP : 1 \; plasmid \gt 3 \; GFP : 1 \; plasmid\)</span></p>
| + | |
| − | | + | |
| − | <p>Therefore, we can use a ratio of 1 uL of the PCR purified GFP solution to 1 uL of the digested plasmid solution.</p>
| + | |
| − | | + | |
| − | <p> </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!-- Add more about the biology of this part here
| + | |
| − | ===Usage and Biology===
| + | |
| | | | |
| − | <!-- -->
| + | 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> |
IP free synthetic non-Aequorea cyan fluorescent protein with ribosome binding site.
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":
The excitation and emission spectra match the specifications on the DNA2.0 website. See 'experience' tab above for spectra.
Sequence and Features
