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<li><p>Even if your electroporation arced, it is possible that you might still have a transformed clone.</p></li> | <li><p>Even if your electroporation arced, it is possible that you might still have a transformed clone.</p></li> | ||
</ul> | </ul> | ||
+ | <h5>Generation of standard curve for plasmid copy number determination </h5> | ||
+ | <h5>Primers used:</h5> | ||
+ | <p>Chromosome gene: dxs (single genome gene - 113).</p> | ||
+ | <p>dxs forward primer: 5’-CGAGAAACTGGCGATCCTTA-3’</p> | ||
+ | <p>dxs reverse primer: 5’-CTTCATCAAGCGGTTTCACA-3’</p> | ||
+ | <br> | ||
+ | <p>Plasmid gene: Specific sequence (189 bp)</p> | ||
+ | <p>Forward primer: 5’-CAGCTCAGAATGCTGGATAGTG-3’</p> | ||
+ | <p>Reverse primer: 5’-CTCAACCTACACATCATCGCAG-3’</p> | ||
+ | <h5>Standard curve generation qPCR </h5> | ||
+ | <p>Standard sample qPCR</p> | ||
+ | <ol> | ||
+ | <li><p>Dilute chromosome and plasmid standard samples to 21ng/uL</p></li> | ||
+ | <li><p> Make a fusion dilution by mixing 3 μL of chromosome standard with 6 μL plasmid plasmid sample and adding water to 20 uL.</p></l<p>(Depends on your standard).</p> | ||
+ | <li><p> Make 6 series of dilutions to obtain 100 – 10-6 of chromosome and plasmid standard dilutions</p></li> | ||
+ | <li><p> For X reactions, make two different mixes using chromosome gene primers and plasmid gene primers:</p></li> | ||
+ | <p> X*6 μL of water </p> | ||
+ | <p>X*1 μL Forward primer 20uM (chromosome and plasmid)</p> | ||
+ | <p> X*1 μL Reverse primer 20uM (chromosome and plasmid)</p> | ||
+ | <p> X*10 μL of Sybr Green master mix</p> | ||
+ | <li><p>First, transfer 18 μL of mix with chromosome primers to first X tubes, then transfer 18 μL of plasmid primers mix to other X tubes (X*2 tubes)</p></li> | ||
+ | <li><p>Add 2 μL of each diluted sample to all the qPCR tubes and gently close the caps.</p></li> | ||
+ | <li><p>Run the reaction.</p></li> | ||
+ | <p>For steps 4-7, increase the volumes by a factor of desired technical replicate numbers.</p> | ||
+ | <p>Avoid direct light source when working with Sybr Green reagent.</p> | ||
+ | <p>The starting concentration of your standard depends on the standard type and copy number of the plasmids counted. </p> | ||
+ | <br> | ||
+ | <p>Cycler conditions </p> | ||
+ | <br> | ||
+ | <table style="width:100%"> | ||
+ | <thead> | ||
+ | <td align='center'>Step</td> | ||
+ | <td align='center'>Time</td> | ||
+ | <td align='center'>Temperature</td> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>PCR initial activation step</td> | ||
+ | <td align='center'>5 min</td> | ||
+ | <td align='center'>95° C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Two-step cycling</td> | ||
+ | <td align='center'></td> | ||
+ | <td align='center'></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Denaturation</td> | ||
+ | <td align='center'>10 s</td> | ||
+ | <td align='center'>95° C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Combined annealing/extension</td> | ||
+ | <td align='center'>30 s</td> | ||
+ | <td align='center'>60° C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Repeat two-step cycling for 35-40 cycles</td> | ||
+ | <td align='center'></td> | ||
+ | <td align='center'></td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <br> | ||
+ | <h5>Standard Curve Generation</h5> | ||
+ | <br> | ||
+ | <p>Data analysis:<p/> | ||
+ | <br> | ||
+ | <ol> | ||
+ | <li><p>Calculate the mass of the DNA fragment used in standard in daltons.</p></li> | ||
+ | <li><p>Gene mass = gene mass in Da * 1 atomic mass unit.</p> | ||
+ | <li><p>Calculate the gene concentration by dividing standard sample concentration by 1 plasmid mass.</p></li> | ||
+ | <li><p>Calculate the gene concentrations for all the serial dillutions.</p></li> | ||
+ | <li><p>Calculate the plasmid copy number in the standard samples the same way.</p></li> | ||
+ | <li><p>Assign the obtained chromosome Ct values to chromosome number in the sample by plotting a graph Chromosome Ct = f(log(chromosome number).</p></li> | ||
+ | <li><p>Assign the obtained plasmid Ct to the the real number of plasmids by plotting plasmid Ct = f(log(plasmid number)).</p></li> | ||
+ | </ol> | ||
+ | <br> | ||
+ | <h5>Copy Number Determination qPCR</h5> | ||
+ | <br> | ||
+ | <p>Lysate standard sample qPCR<p/> | ||
+ | <br> | ||
+ | <ol> | ||
+ | <li><p>Inoculate a single colony into 5 mL of liquid LB medium with corresponding antibiotic and incubate in the shaker at 37oC.</p></li> | ||
+ | <li><p>After 14-16 h of growth, transfer 100 μL of suspended cells to fresh 5 mL of liquid LB medium with corresponding antibiotic and incubate at 37oC untill the OD600 reaches 0.7-0.8.</p> | ||
+ | <li><p>Spin down a suspended 1 mL of cells of 0.7 OD600 at 8.0g for 15 min.</p></li> | ||
+ | <p>(Growth conditions are specified at the end of the protocol).</p> | ||
+ | <li><p>Remove the medium and resuspend the cell pellet in 1 mL of PBS.</p></li> | ||
+ | <li><p>Spin down the suspended of cells at 8.0g for 15 min.</p></li> | ||
+ | <li><p>Repeat steps 2 and 3.</p></li> | ||
+ | <li><p>Completely remove PBS from the cell pellet.</p></li> | ||
+ | <li><p>Incubate cells at 95o C for 10 min.</p></li> | ||
+ | <li><p>Store cells at -20o C for 10 min.</p></li> | ||
+ | <li><p>Completely resuspend dry cell pellet in 100 μL of water by pipetting. Then vortex for 30s and spin down.</p></li> | ||
+ | <li><p>Make an initial dilution by transferring 10 μL of resuspended cell to 40 μL of water. Pipet carefully vortex for 30s and spin down.</p></li> | ||
+ | <li><p>Make a second dilution by transferring 10 μL of to 90 μL of water. Pipet carefully vortex for 30s and spin down.</p></li> | ||
+ | <li><p>For X reactions, make two different mixes using chromosome gene and plasmid gene primers:</p></li> | ||
+ | <p>X*6 μL of water</p> | ||
+ | <p>X*1 μL Forward primer 20uM</p> | ||
+ | <p>X*1 μL Reverse primer 20uM</p> | ||
+ | <p>X*10 μL of SYBR Green</p> | ||
+ | <li><p>First, transfer 18 μL of mix with chromosome primers to first X tubes, then transfer | ||
+ | 18 μL of plasmid primers mix to other X tubes (X*2 tubes)</p></li> | ||
+ | <li><p>Add 2 μL of each diluted sample to the tubes.</p></li> | ||
+ | <li><p>Gently close the caps.</p></li> | ||
+ | <li><p>Run the reaction. </p></li> | ||
+ | <p>For steps 13-15, increase the volumes by a factor of desired technical replicate numbers.</p> | ||
+ | <br> | ||
+ | </ol> | ||
+ | <table style="width:100%"> | ||
+ | <thead> | ||
+ | <td align='center'>Step</td> | ||
+ | <td align='center'>Time</td> | ||
+ | <td align='center'>Temperature</td> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>PCR initial activation step</td> | ||
+ | <td align='center'>5 min</td> | ||
+ | <td align='center'>95° C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Two-step cycling</td> | ||
+ | <td align='center'></td> | ||
+ | <td align='center'></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Denaturation</td> | ||
+ | <td align='center'>10 s</td> | ||
+ | <td align='center'>95° C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Combined annealing/extension</td> | ||
+ | <td align='center'>30 s</td> | ||
+ | <td align='center'>60° C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Repeat two-step cycling for 35-40 cycles</td> | ||
+ | <td align='center'></td> | ||
+ | <td align='center'></td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <br> | ||
+ | <h5>Data Analysis</h5> | ||
+ | <br> | ||
+ | <ol> | ||
+ | <li><p>By using the equation from standard curve that relates plasmid Ct value to real plasmid number calculate the plasmid number in the sample. </p></li> | ||
+ | <li><p>By using the equation that relates chromosome Ct to real chromosome number calculate the number of chromosomes in the sample. </p> | ||
+ | <li><p>Chromosome number = cell number. Therefore, by dividing the obtained plasmid number by chromosome number we can find the plasmid per cell number. </p></li> | ||
+ | </ol> | ||
<p>[1] Q. Tu, J. Yin, J. Fu, J. Herrmann, Y. Li, Y. Yin, A. F. Stewart, R. Müller and Y. Zhang, 2016, 6, 24648.</p> | <p>[1] Q. Tu, J. Yin, J. Fu, J. Herrmann, Y. Li, Y. Yin, A. F. Stewart, R. Müller and Y. Zhang, 2016, 6, 24648.</p> | ||
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Revision as of 03:15, 2 November 2017
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