Team:Macquarie Australia/Notebook
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- | <h4>Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene: </h4> | + | <h4>Fwd and Rvs primers for amplification of the full length <i> A. tumefaciens </i> BphP gene: </h4> |
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+ | <h4>Fwd and Rvs primers for amplification of the full length <i> A. tumefaciens </i>BphP gene for insertion in the operon <u>BEFORE</u> the HO gene: | ||
+ | </h4> | ||
+ | |||
+ | <div align="center"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Primer name</th> | ||
+ | <th>Primer Sequence</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>F2 (AT-BHO-F)</td> | ||
+ | <td>5’- AAG GAG ATA TAC ATA TGA TGA GTT CAC ATA CGC CG – 3’</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R3 (AT-BHO-R)</td> | ||
+ | <td> 5’- AAG TTG ACA CTC ATA TGA GCC CTC CTT TCA GGC – 3’</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | <p> </div> | ||
+ | |||
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+ | #instructions{ color: #80 00 00;text-align: center; font-weight: normal; font-size: large; padding: 10px;} | ||
+ | #box{text-align: center; font-weight: bold; font-size: large; color: #80 00 00; padding: 15px;} | ||
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+ | body {background-color:#A11F37} | ||
+ | </style> | ||
+ | </head> | ||
+ | <body> | ||
+ | <div id="sidebar" > | ||
+ | <ul id="navigation"> | ||
+ | |||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia">Home</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Team">Team</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Project">Project</li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Parts">Parts Submitted to the Registry</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Modeling">Modeling</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Notebook">Notebook</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Safety">Safety</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/aboutus">About Us</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/References">References</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Just for fun!">Just for fun!</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Acknowledgements">Acknowledgements</a></li> | ||
+ | <li><a href="https://igem.org/Team.cgi?year=2010">Official Team Profile</a></li> | ||
+ | </ul> | ||
+ | |||
+ | </div> | ||
+ | <div id="body"> | ||
+ | <h1><font color="#47484c">PROJECT LAB BOOK <p> | ||
+ | <hr> | ||
+ | |||
+ | Welcome to the Macquarie University project lab book page! <p> | ||
+ | |||
+ | Here you will find a day-by-day account of our triumphs and failures.</font></h1> </hr> | ||
+ | |||
+ | <p><p> | ||
+ | |||
+ | |||
+ | <center><h2> A day-by-day progress for <i> Agrobacterium Tumefaciens </i> Bacteriophytochrome </h2> </center> | ||
+ | <p><p> | ||
+ | <big> | ||
+ | 20th August 2010 <p> | ||
+ | |||
+ | Genomic DNA extraction <p> </big> | ||
+ | |||
+ | <menu> | ||
+ | <li type="disc"> | ||
+ | |||
+ | <i> A. tumefaciens </i> genomic DNA extracted using the Invitrogen PureLinkTM Genomic DNA Purification kit as per the manufacturer’s protocols. </li> <li type="disc"> <p> | ||
+ | The extractions were run on a GelRed stained 1% agarose gel and photo taken for visualization (see below). </li> <p> <li type="disc"> | ||
+ | A NanoDrop spectrophotometer reading was also recorded to check the quality of the extracted genomic DNA. <p> </li> <li type="disc"> | ||
+ | The extraction was successful for all A. tumefaciens cell lysate samples (labeled DNA1.1, DNA1.2, DNA2 (See figure below) <p> </li> | ||
+ | |||
+ | <p><h3><i><b>A. tumefaciens </i> genomic DNA extraction agarose results: <p> </b> </h3> | ||
+ | |||
+ | All four DNA samples show a smear of gDNA. Because the samples were treated with RNase there is no band indicative of RNA visible = SUCCESS! <p> | ||
+ | |||
+ | |||
+ | |||
+ | <h4> <center>***** Results of A. tumefaciens genomic DNA extraction ***</h4></center></big> <p> | ||
+ | |||
+ | |||
+ | <b>Figure 1. </b> GelRed stained 1% agarose gel of genomic DNA extraction from A. tumefaciens.<p> | ||
+ | In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.1 flow through, lane 3 is the DNA2.2 flow through, lane 4 is the DNA2.1 flow through and lane 4 in the DNA2.2 flowthrough. All four samples show a smear that is indicative of genomic DNA. <u> The extraction has been successful. </u> | ||
+ | |||
+ | <h4>Nanodrop absorbance readings: </h4> | ||
+ | |||
+ | <div align="center"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Genomic DNA sample</th> | ||
+ | <th>260/280 OD ratio</th> | ||
+ | <th>Concentration (ng/mL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA1.1</td> | ||
+ | <td>1.88</td> | ||
+ | <td>351.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA1.2</td> | ||
+ | <td>1.98</td> | ||
+ | <td>203.1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA2.1</td> | ||
+ | <td>2.14</td> | ||
+ | <td>738.9</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA2.2</td> | ||
+ | <td>2.16</td> | ||
+ | <td>709.7</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | <p> </div> | ||
+ | Overall, the Nanodrop readings show that we have obtained good DNA concentration with minimal protein contamination <p>= <u>SUCCESS! </u><p> | ||
+ | |||
+ | |||
+ | <p><p> | ||
+ | <big><hr> | ||
+ | 27th August 2010 <p> | ||
+ | |||
+ | Primer design <p> </big> </hr> | ||
+ | |||
+ | |||
+ | <li type="disc"> | ||
+ | Various primers were designed manually and using Primer 3 Software package for PCR amplification.</li> <p> | ||
+ | <li type="disc"> | ||
+ | The primers were ordered and supplied through XXXXXXXXXXXXXXXXX.</li><p> | ||
+ | <li type="disc"> | ||
+ | There was an array of various primers ordered for amplification of different products. The details of the primers are described below. </li><p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4>Fwd and Rvs primers for amplification of the full length <i> A. tumefaciens </i> BphP gene: </h4> | ||
+ | |||
+ | <div align="center"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Primer name</th> | ||
+ | <th>Primer Sequence</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>F1 (AT-FWD-1)</td> | ||
+ | <td>5’-ATG AGT TCA CAT ACG CCG-3’</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R1 (AT-RVS-1)</td> | ||
+ | <td>5’-TCA GGC AAT TTT TTC CTC-3’</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | <p> </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4> | ||
+ | Fwd and Rvs primers for amplification of the full length <i>A. tumefaciens </i> BphP gene for insertion in the operon <u>AFTER </u>the HO gene in the operon: | ||
+ | |||
+ | </h4> | ||
+ | |||
+ | <div align="center"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Primer name</th> | ||
+ | <th>Primer Sequence</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>F4 (AT-AHO-F)</td> | ||
+ | <td>5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG – 3’</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R2 (AT-AHO-R)</td> | ||
+ | <td> 5’- GTT AGC CGG ATC CTC AGG CAA TTT TTT CCT – 3’</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | <p> </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4> | ||
+ | Fwd and Rvs primers for amplification of the full length <i>A. tumefaciens </i> BphP gene for insertion in the operon <u>AFTER </u>the HO gene as well as the addition of a ribosome binding site or Shine Delgano sequence: | ||
+ | |||
+ | </h4> | ||
+ | |||
+ | <div align="center"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Primer name</th> | ||
+ | <th>Primer Sequence</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>F3 (AT-FWD-RBS)</td> | ||
+ | <td> 5’- AGG AGG GCT ATG AGT TCA CAT ACG CCG -3’</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | <p> </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
Revision as of 01:17, 14 October 2010
PROJECT LAB BOOK
Welcome to the Macquarie University project lab book page!
Here you will find a day-by-day account of our triumphs and failures.
A day-by-day progress for Agrobacterium Tumefaciens Bacteriophytochrome
20th August 2010
Genomic DNA extraction