Team:Macquarie Australia/Notebook
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<li><a href="https://2010.igem.org/Team:Macquarie_Australia">Home</a></li> | <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/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/Project">Project</a></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/Parts">Parts Submitted to the Registry</a></li> | ||
- | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/ | + | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Glossary">Glossary</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/humanpractice">Human practice</a></li> |
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Notebook">Notebook 1: <i>Agrobacterium Tumefaciens</i> | ||
+ | </a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Notebook2">Notebook 2: <i>Deinococcus Radiodurans</i> | ||
+ | </a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Notebook3">Notebook 3: Cloning | ||
+ | </a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Macquarie_Australia/Protocols and Other Methods">Protocols and Other Methods</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/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/aboutus">About Us</a></li> | ||
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</div> | </div> | ||
<div id="body"> | <div id="body"> | ||
- | < | + | <h2><font color="#47484c"><Center>PROJECT LAB BOOK</Center> <p> |
<hr> | <hr> | ||
- | + | <Center> | |
Welcome to the Macquarie University project lab book page! <p> | 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></ | + | Here you will find a day-by-day account of our triumphs and failures.</Center></font></h2> </hr> |
<p><p> | <p><p> | ||
+ | <center> <a target='_blank' title='ImageShack - Image And Video Hosting' href='http://img687.imageshack.us/i/10176886.png/'><img src='http://img687.imageshack.us/img687/2149/10176886.png' border='0'/></a> | ||
+ | |||
+ | </a> </center> | ||
+ | |||
+ | </a> | ||
+ | <hr><p> | ||
<center><h2> A day-by-day progress for <i> Agrobacterium Tumefaciens </i> Bacteriophytochrome </h2> </center> | <center><h2> A day-by-day progress for <i> Agrobacterium Tumefaciens </i> Bacteriophytochrome </h2> </center> | ||
- | <p><p> | + | <p></p><p> |
- | <big> | + | |
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+ | <hr> | ||
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+ | <big><b> | ||
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20th August 2010 <p> | 20th August 2010 <p> | ||
- | Genomic DNA extraction <p> </big> | + | Genomic DNA extraction <p> </big> </b> |
<menu> | <menu> | ||
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The extractions were run on a GelRed stained 1% agarose gel and photo taken for visualization (see below). </li> <p> <li type="disc"> | 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"> | 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> | + | The extraction was successful for all A. tumefaciens cell lysate samples (labeled DNA1.1, DNA1.2, DNA2.1, DNA2.2 (See figure below) <p> </li> |
<p><h3><i><b>A. tumefaciens </i> genomic DNA extraction agarose results: <p> </b> </h3> | <p><h3><i><b>A. tumefaciens </i> genomic DNA extraction agarose results: <p> </b> </h3> | ||
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- | <h4> <center> | + | <h4> <center><h3> Figure 1. Results of <i> A. tumefaciens </i> genomic DNA extraction </h4></center></big> <p></h3> |
+ | |||
+ | <center> | ||
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+ | <a target='_blank' title='ImageShack - Image And Video Hosting' href='http://img191.imageshack.us/i/gel1.png/'><img src='http://img191.imageshack.us/img191/5232/gel1.png' border='0'/></a> | ||
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+ | </a> </center> | ||
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<b>Figure 1. </b> GelRed stained 1% agarose gel of genomic DNA extraction from A. tumefaciens.<p> | <b>Figure 1. </b> GelRed stained 1% agarose gel of genomic DNA extraction from A. tumefaciens.<p> | ||
- | In | + | In lane 1 there is a 1kb ladder. In lane 2 is the DNA1.1 flow through, lane 3 is the DNA1.2 flow through, lane 4 is the DNA2.1 flow through and lane 5 is 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> | <h4>Nanodrop absorbance readings: </h4> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
</center> | </center> | ||
- | <p> </ | + | <p> </center> |
Overall, the Nanodrop readings show that we have obtained good DNA concentration with minimal protein contamination <p>= <u>SUCCESS! </u><p> | Overall, the Nanodrop readings show that we have obtained good DNA concentration with minimal protein contamination <p>= <u>SUCCESS! </u><p> | ||
<p><p> | <p><p> | ||
- | <big><hr> | + | <big><hr><b> |
27th August 2010 <p> | 27th August 2010 <p> | ||
- | Primer design <p> </big> </hr> | + | Primer design <p> </big> </hr></b> |
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Various primers were designed manually and using Primer 3 Software package for PCR amplification.</li> <p> | Various primers were designed manually and using Primer 3 Software package for PCR amplification.</li> <p> | ||
<li type="disc"> | <li type="disc"> | ||
- | The primers were ordered and supplied through | + | The primers were ordered and supplied through Integrated DNA Technologies.</li><p> |
<li type="disc"> | <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> | There was an array of various primers ordered for amplification of different products. The details of the primers are described below. </li><p> | ||
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<h4>Fwd and Rvs primers for amplification of the full length <i> A. tumefaciens </i> BphP gene: </h4> | <h4>Fwd and Rvs primers for amplification of the full length <i> A. tumefaciens </i> BphP gene: </h4> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td> (AT-FWD-1)</td> |
<td>5’-ATG AGT TCA CAT ACG CCG-3’</td> | <td>5’-ATG AGT TCA CAT ACG CCG-3’</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>(AT-RVS-1)</td> |
<td>5’-TCA GGC AAT TTT TTC CTC-3’</td> | <td>5’-TCA GGC AAT TTT TTC CTC-3’</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</center> | </center> | ||
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</h4> | </h4> | ||
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<table> | <table> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>(AT-BHO-F)</td> |
<td>5’- AAG GAG ATA TAC ATA TGA TGA GTT CAC ATA CGC CG – 3’</td> | <td>5’- AAG GAG ATA TAC ATA TGA TGA GTT CAC ATA CGC CG – 3’</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>(AT-BHO-R)</td> |
<td> 5’- AAG TTG ACA CTC ATA TGA GCC CTC CTT TCA GGC – 3’</td> | <td> 5’- AAG TTG ACA CTC ATA TGA GCC CTC CTT TCA GGC – 3’</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</center> | </center> | ||
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</h4> | </h4> | ||
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<table> | <table> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>(AT-AHO-F)</td> |
<td>5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG – 3’</td> | <td>5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG – 3’</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>(AT-AHO-R)</td> |
<td> 5’- GTT AGC CGG ATC CTC AGG CAA TTT TTT CCT – 3’</td> | <td> 5’- GTT AGC CGG ATC CTC AGG CAA TTT TTT CCT – 3’</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
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</h4> | </h4> | ||
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<table> | <table> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>(AT-FWD-RBS)</td> |
<td> 5’- AGG AGG GCT ATG AGT TCA CAT ACG CCG -3’</td> | <td> 5’- AGG AGG GCT ATG AGT TCA CAT ACG CCG -3’</td> | ||
</tr> | </tr> | ||
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</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
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<li type="disc">The reaction mastermix for the initial PCR was set up as per the following recipe (per sample): </li> | <li type="disc">The reaction mastermix for the initial PCR was set up as per the following recipe (per sample): </li> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
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<tr> | <tr> | ||
- | <td> | + | <td>Gibco H2O</td> |
<td> 13.75</td> | <td> 13.75</td> | ||
</tr> | </tr> | ||
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</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
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Different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following) </li> <li type="disc"> | Different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following) </li> <li type="disc"> | ||
Not all possible primer combinations were used due to limitations on the amount of polymerase enzyme available to us </li><li type="disc"> | Not all possible primer combinations were used due to limitations on the amount of polymerase enzyme available to us </li><li type="disc"> | ||
- | The PCR products were run on a 1.2% GelRed stained agarose gel for visualisation (see picture of gel below). </li> | + | The PCR products were run on a 1.2% GelRed post-stained agarose gel for visualisation (see picture of gel below). </li> |
<p><p> | <p><p> | ||
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<h4>Experimental Design – Primer combinations: </h4> | <h4>Experimental Design – Primer combinations: </h4> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
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<tr> | <tr> | ||
<td>DNA1.2</td> | <td>DNA1.2</td> | ||
- | <td> | + | <td> (AT-FWD-1)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td>(AT-FWD-1)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DNA1.2</td> | <td>DNA1.2</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DNA2.2 </td> | <td>DNA2.2 </td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA1.2</td> | <td> DNA1.2</td> | ||
- | <td> | + | <td>(AT-FWD-RBS) </td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td>(AT-FWD-RBS)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> SSH20 (negative control)</td> | <td> SSH20 (negative control)</td> | ||
- | <td> | + | <td>(AT-FWD-1)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
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</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
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<li type="disc"> | <li type="disc"> | ||
- | A band was seen in lane | + | A band was seen in lane 9. This lane was using the primer and DNA template combinations of DNA2.2, (AT-FWD-RBS) and (AT-RVS-1) primers <p>= SUCCESS!!! </li> |
- | + | ||
- | + | ||
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- | < | + | <h3> <center> Figure 2. Results of the initial PCR </h3></center></big> <p> |
- | <b>Figure 2.</b> GelRed stained 1.2% agarose gel of initial PCR of <i>A. tumefaciens </i> using various primers. <p> | + | <center> |
- | In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.2 template with | + | |
+ | |||
+ | |||
+ | <a target='_blank' title='ImageShack - Image And Video Hosting' href='http://img411.imageshack.us/i/gel2.png/'><img src='http://img411.imageshack.us/img411/8184/gel2.png' border='0'/></a> | ||
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+ | </a></center> | ||
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+ | <b>Figure 2.</b> GelRed post-stained 1.2% agarose gel of initial PCR of <i>A. tumefaciens </i> using various primers. <p> | ||
+ | In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.2 template with (AT-FWD-1) and (AT-RVS-1)primer pair. <p>In lane 3 is the DNA2.2 template with (AT-FWD-1) and (AT-RVS-1)primer pair. In lane 4 is the DNA1.2 template with (AT-BHO-F) and (AT-RVS-1)primer pair. In lane 5 is the DNA2.2 template with the (AT-BHO-F) and (AT-RVS-1)primer pair. In lanes 6 and 7 there is nothing loaded as the wells were damaged. In lane 8 there is the DNA1.2 template with (AT-FWD-RBS) and (AT-RVS-1)primer pair. In lane 9 there is the DNA2.2 template with the (AT-FWD-RBS)and (AT-RVS-1)primer pair and in lane 10 there is the ssH2O negative control. A product is seen in lane 9 – this is the DNA2.2 template with the (AT-FWD-RBS)and (AT-RVS-1)primers! <u>This means that we have a <i> A. tumefaciens bacteriophytochrome </i> product with a ribosome binding sight inserted. </u> <p> | ||
</u> | </u> | ||
<hr> | <hr> | ||
- | <big> | + | <big> <b> |
+ | |||
30th August 2010 <p> | 30th August 2010 <p> | ||
- | PCR Optimization<p> </big> </hr> | + | PCR Optimization<p> </big> </hr> </b> |
- | <li type="disc"> Now that we have a product obtained by the initial PCR it is time to optimize the PCR conditions using the successful DNA2.2 PCR product obtained from last week and the original DNA2.2 template <li> | + | <li type="disc"> Now that we have a product obtained by the initial PCR it is time to optimize the PCR conditions using the successful DNA2.2 PCR product obtained from last week and the original DNA2.2 template </li> |
<li type="disc"> The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li> | <li type="disc"> The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
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<tr> | <tr> | ||
- | <td> | + | <td>Gibco H2O</td> |
<td> 13.75</td> | <td> 13.75</td> | ||
</tr> | </tr> | ||
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</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
<h4> The PCR program was set up as per the following: </h4> | <h4> The PCR program was set up as per the following: </h4> | ||
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<li type="disc">Again, different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following) </li> | <li type="disc">Again, different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following) </li> | ||
<li type="disc">Now that we don’t have a limited enzyme supply, even more primer combinations can be used!!</li> | <li type="disc">Now that we don’t have a limited enzyme supply, even more primer combinations can be used!!</li> | ||
- | <li type="disc">The PCR products were run on a GelRed stained 2% agarose gel using a 100bp ladder for visualization</li> | + | <li type="disc">The PCR products were run on a GelRed post-stained 2% agarose gel using a 100bp ladder for visualization</li> |
<li type="disc">All primer combinations tested worked with bands visible in each lane! The strange band pattern seen in lane 6 is most probably due to non-specific binding.</li> | <li type="disc">All primer combinations tested worked with bands visible in each lane! The strange band pattern seen in lane 6 is most probably due to non-specific binding.</li> | ||
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<h4>Experimental Design – Primer combinations: </h4> | <h4>Experimental Design – Primer combinations: </h4> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
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<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td> (AT-FWD-1)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PCR product (from DNA2.2)</td> | <td> PCR product (from DNA2.2)</td> | ||
- | <td> | + | <td>(AT-FWD-1)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PCR product (from DNA2.2)</td> | <td> PCR product (from DNA2.2)</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td>(AT-FWD-RBS) </td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PCR product (from DNA2.2)</td> | <td> PCR product (from DNA2.2)</td> | ||
- | <td> | + | <td>(AT-FWD-RBS)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td>(AT-FWD-1)</td> |
- | <td> | + | <td> (AT-AHO-R)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PCR product (from DNA2.2)</td> | <td> PCR product (from DNA2.2)</td> | ||
- | <td> | + | <td>(AT-FWD-1)</td> |
- | <td> | + | <td> (AT-AHO-R)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-AHO-R)</td> |
</tr> | </tr> | ||
Line 575: | Line 610: | ||
<tr> | <tr> | ||
<td> PCR product (from DNA2.2)</td> | <td> PCR product (from DNA2.2)</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-AHO-R)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> DNA2.2</td> | <td> DNA2.2</td> | ||
- | <td> | + | <td> (AT-FWD-RBS)</td> |
- | <td> | + | <td> (AT-AHO-R)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PCR product (from DNA2.2)</td> | <td> PCR product (from DNA2.2)</td> | ||
- | <td> | + | <td> (AT-FWD-RBS)</td> |
- | <td> | + | <td> (AT-AHO-R)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> ssH2O (-) control </td> | <td> ssH2O (-) control </td> | ||
- | <td> | + | <td> (AT-FWD-1)</td> |
- | <td> | + | <td> (AT-RVS-1)</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
Line 606: | Line 641: | ||
- | < | + | <h3> <center> Figure 3. PCR optimization results </h3></center></big> <p> |
+ | <center> | ||
+ | <a target='_blank' title='ImageShack - Image And Video Hosting' href='http://img146.imageshack.us/i/gel3w.png/'><img src='http://img146.imageshack.us/img146/9223/gel3w.png' border='0'/></a> | ||
- | <b>Figure 3. </b> GelRed stained 2% agarose gel of optimized PCR of <i> A. tumefaciens bacteriophytochrome </i> using various primers. In lanes 1 and 15 there is a 100bp ladder. In lane 2 is the DNA2.2 sample with | + | </a> |
+ | |||
+ | |||
+ | |||
+ | |||
+ | </center> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <b>Figure 3. </b> GelRed post-stained 2% agarose gel of optimized PCR of <i> A. tumefaciens bacteriophytochrome </i> using various primers. In lanes 1 and 15 there is a 100bp ladder. In lane 2 is the DNA2.2 sample with (AT-FWD-1) and (AT-RVS-1)primer pair. In lane 3 there is PCR product template (from DNA2.2 sample) with (AT-FWD-1)and (AT-RVS-1)primer pair. In lane 4 there is DNA sample with (AT-BHO-F) and (AT-RVS-1)primer pair. In lane 5 is the PCR product (from DNA2.2 sample) with (AT-BHO-F)and (AT-RVS-1)primer pair. In lane 6 there is DNA2.2 sample with (AT-FWD-RBS)and (AT-RVS-1)primer pair. In lane 7 there is PCR product template (from DNA2.2 sample) with (AT-FWD-RBS)and (AT-RVS-1)primer pair. In lane 8 there is DNA2.2 sample with (AT-FWD-1) and (AT-AHO-R)primer pairs. In lane 9 there is PCR product template (from DNA2.2 sample) with (AT-FWD-1) and (AT-AHO-R)primer pairs. In lane 10 there is DNA2.2 sample with (AT-BHO-F) and (AT-AHO-R)primer pairs. In lane 11 there is PCR product (from DNA2.2 sample) with (AT-BHO-F)and (AT-AHO-R)primer pair. In lane 12 there is DNA2.2 sample with (AT-BHO-F)and (AT-AHO-R)primer pair. In lane 13 there is PCR product (from DNA2.2 sample) with (AT-FWD-RBS)and (AT-AHO-R)primer pair. In lane 14 there is ssH2O negative control with (AT-FWD-1)and (AT-RVS-1)primer pair. <u> All the lanes worked. There is a weird result in lane 7 which is probably due to non-specific binding. = SUCCESS! <p> | ||
</u> | </u> | ||
<hr> | <hr> | ||
- | <big> | + | <big> <b> |
+ | |||
3rd September 2010 <p> | 3rd September 2010 <p> | ||
- | PCR Optimization (using Gradient PCR)<p> </big> </hr> <p> | + | PCR Optimization (using Gradient PCR)<p> </big> </hr> <p> </b> |
Line 622: | Line 670: | ||
<li type="disc">We will be inserting the two genes in two different orientations in the operon </li> | <li type="disc">We will be inserting the two genes in two different orientations in the operon </li> | ||
<li type="disc">We will achieve this using two different primer pairs </li> | <li type="disc">We will achieve this using two different primer pairs </li> | ||
- | <ul type="circle"> <li>The first primer pair is | + | <ul type="circle"> <li>The first primer pair is (AT-BHO-F) with (AT-BHO-R) [this will insert the bacteriophytchrome gene BEFORE the heme oxygenase gene in the operon] </li> |
- | <li> The second primer pair is | + | <li> The second primer pair is (AT-AHO-F) with (AT-AHO-R) [this will insert the bacteriophytochrome gene AFTER the heme oxygenase gene in the operon] </ul> </li> |
<li type="disc">A technique called gradient PCR will be used here. This PCR includes different annealing temperatures so that the optimum annealing temperature for the primers can be determined. </li> | <li type="disc">A technique called gradient PCR will be used here. This PCR includes different annealing temperatures so that the optimum annealing temperature for the primers can be determined. </li> | ||
<li type="disc">This should also result in reduced non-specific binding that was observed in the previous PCR result </li> | <li type="disc">This should also result in reduced non-specific binding that was observed in the previous PCR result </li> | ||
Line 630: | Line 678: | ||
<li type="disc">The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li> | <li type="disc">The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 638: | Line 686: | ||
<tr> | <tr> | ||
- | <td> | + | <td>Gibco H2O</td> |
<td> 13.75</td> | <td> 13.75</td> | ||
</tr> | </tr> | ||
Line 673: | Line 721: | ||
</table> | </table> | ||
</center> | </center> | ||
- | <p> </ | + | <p> </b> |
Line 697: | Line 745: | ||
- | <li type="disc">The PCR products were run on a GelRed stained 2% agarose gel using a 1kb ladder for visualization </li> | + | <li type="disc">The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization </li> |
- | <li type="disc">The products were run on a GelRed stained 2% agarose gel for visualization </li> | + | <li type="disc">The products were run on a GelRed post-stained 2% agarose gel for visualization </li> |
- | <li type="disc"> | + | <li type="disc">No products were observed </li> |
Line 710: | Line 758: | ||
<h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | <h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 724: | Line 772: | ||
<tr> | <tr> | ||
- | <td> | + | <td>(AT-BHO-F)</td> |
- | <td> | + | <td> (AT-BHO-R)</td> |
<td> 57.1 </td> | <td> 57.1 </td> | ||
<td> 58.7 </td> | <td> 58.7 </td> | ||
Line 734: | Line 782: | ||
<tr> | <tr> | ||
- | <td> | + | <td> (AT-AHO-F) </td> |
- | <td> | + | <td>(AT-AHO-R)</td> |
<td> 57.1</td> | <td> 57.1</td> | ||
<td> 58.7</td> | <td> 58.7</td> | ||
Line 748: | Line 796: | ||
</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
Line 756: | Line 804: | ||
- | < | + | <h3> <center> Figure 4. PCR Optimisation (gradient PCR) results</h3></center></big> <p> |
+ | |||
+ | |||
+ | <center> | ||
+ | |||
+ | |||
+ | |||
+ | <a target='_blank' title='ImageShack - Image And Video Hosting' href='http://img43.imageshack.us/i/gel4.png/'><img src='http://img43.imageshack.us/img43/8279/gel4.png' border='0'/></a> | ||
+ | |||
+ | </a> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </center> | ||
+ | |||
Line 762: | Line 829: | ||
- | + | No products were observed. The anticipated product size was between 1.6kb and 3kb. <u>The gel was over run but the only products that were over run were probably primer dimers, which are less than 300bp in size. = FAIL!</u> | |
</u> | </u> | ||
- | <hr> | + | <hr> <b> |
+ | |||
<big> | <big> | ||
7th September 2010 <p> | 7th September 2010 <p> | ||
- | PCR Optimization (using Gradient PCR) <p> </big> </hr> <p> | + | PCR Optimization (using Gradient PCR) <p> </big> </hr> <p> </font></b> |
Line 788: | Line 856: | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 796: | Line 864: | ||
<tr> | <tr> | ||
- | <td> | + | <td>Gibco H2O</td> |
<td> 13.75</td> | <td> 13.75</td> | ||
</tr> | </tr> | ||
Line 831: | Line 899: | ||
</table> | </table> | ||
</center> | </center> | ||
- | <p> </ | + | <p> </b> |
<h4> The PCR program was set up as per the following: </h4> | <h4> The PCR program was set up as per the following: </h4> | ||
Line 851: | Line 919: | ||
- | <li type="disc">The PCR products were run on a GelRed stained 2% agarose gel using a 1kb ladder for visualization </li> | + | <li type="disc">The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization </li> |
- | <u><li type="disc"> | + | <u><li type="disc">There was no amplification observed in any of the lanes so the picture of this gel is not included = FAIL! </li> </u> |
Line 859: | Line 927: | ||
<h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | <h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 873: | Line 941: | ||
<td> PCR product (from DNA2.2) </td> | <td> PCR product (from DNA2.2) </td> | ||
<td> 1:100</td> | <td> 1:100</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-BHO-R)</td> |
<td> 60 </td> | <td> 60 </td> | ||
Line 883: | Line 951: | ||
<td> PCR product (from DNA2.2) </td> | <td> PCR product (from DNA2.2) </td> | ||
<td> 1:200</td> | <td> 1:200</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-BHO-R)</td> |
<td> 60</td> | <td> 60</td> | ||
</tr> | </tr> | ||
Line 891: | Line 959: | ||
<td> PCR product (from DNA2.2) </td> | <td> PCR product (from DNA2.2) </td> | ||
<td> 1:100</td> | <td> 1:100</td> | ||
- | <td> | + | <td> (AT-AHO-F) </td> |
- | <td> | + | <td> (AT-AHO-R) </td> |
<td> 60 </td> | <td> 60 </td> | ||
</tr> | </tr> | ||
Line 899: | Line 967: | ||
<td> PCR product (from DNA2.2)</td> | <td> PCR product (from DNA2.2)</td> | ||
<td>1:200</td> | <td>1:200</td> | ||
- | <td> | + | <td> (AT-AHO-F) </td> |
- | <td> | + | <td> (AT-AHO-R) </td> |
<td> 60</td> | <td> 60</td> | ||
</tr> | </tr> | ||
Line 907: | Line 975: | ||
<td> PCR product (from DNA2.2) </td> | <td> PCR product (from DNA2.2) </td> | ||
<td> 1:100</td> | <td> 1:100</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-BHO-R) </td> |
<td> 65 </td> | <td> 65 </td> | ||
</tr> | </tr> | ||
Line 916: | Line 984: | ||
<td> PCR product (from DNA2.2) </td> | <td> PCR product (from DNA2.2) </td> | ||
<td> 1:200</td> | <td> 1:200</td> | ||
- | <td> | + | <td> (AT-BHO-F)</td> |
- | <td> | + | <td> (AT-BHO-R) </td> |
<td> 65 </td> | <td> 65 </td> | ||
</tr> | </tr> | ||
Line 925: | Line 993: | ||
<td> PCR product (from DNA2.2) </td> | <td> PCR product (from DNA2.2) </td> | ||
<td> 1:100</td> | <td> 1:100</td> | ||
- | <td> | + | <td> (AT-AHO-F) </td> |
- | <td> | + | <td> (AT-AHO-R) </td> |
<td> 65 </td> | <td> 65 </td> | ||
</tr> | </tr> | ||
Line 934: | Line 1,002: | ||
<td> PCR product (from DNA2.2) </td> | <td> PCR product (from DNA2.2) </td> | ||
<td>1:200</td> | <td>1:200</td> | ||
- | <td> | + | <td> (AT-AHO-F) </td> |
- | <td> | + | <td> (AT-AHO-R)</td> |
<td> 65 </td> | <td> 65 </td> | ||
</tr> | </tr> | ||
Line 942: | Line 1,010: | ||
</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
<hr> | <hr> | ||
- | <big> | + | <big> <b> |
10th September 2010 <p> | 10th September 2010 <p> | ||
- | PCR Optimization (repeated)<p> </big> </hr> <p> | + | PCR Optimization (repeated)<p> </big> </hr> <p> </font></b> |
<li type="disc">As we were having trouble with one of the primers annealing we attempted a PCR technique that required a different PCR program to be set </li> | <li type="disc">As we were having trouble with one of the primers annealing we attempted a PCR technique that required a different PCR program to be set </li> | ||
- | <li type="disc">The | + | <li type="disc">The (AT-AHO-F)– (AT-AHO-R)primer pair were run on the normal PCR program being used for all other PCR’s as these primers were annealing properly </li> |
- | <li type="disc">The primer pair that we were having difficulty with ( | + | <li type="disc">The primer pair that we were having difficulty with ((AT-BHO-F)-(AT-BHO-R)) required a special PCR program. This would allow for the (AT-BHO-R)primer to anneal to the template first for single stranded amplification and then the (AT-BHO-F)primer to bind later. <p></li> |
<li type="disc">The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li> | <li type="disc">The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li> | ||
- | < | + | <center> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 968: | Line 1,036: | ||
<tr> | <tr> | ||
- | <td> | + | <td>Gibco H2O</td> |
<td> 13.75</td> | <td> 13.75</td> | ||
</tr> | </tr> | ||
Line 1,003: | Line 1,071: | ||
</table> | </table> | ||
</center> | </center> | ||
- | <p | + | <p> |
Line 1,032: | Line 1,100: | ||
- | <h4> The <u> second </u> PCR program was set up as per the following: </h4> | + | <h4> The <u> second </u> PCR program was set up as per the following (this was for the amplification of product using the (AT-FWD-RBS) and (AT-AHO-R) primer pair): </h4> |
<ol> | <ol> | ||
Line 1,041: | Line 1,109: | ||
<li>72˚C for 2 minutes & 30 seconds</li> <p> | <li>72˚C for 2 minutes & 30 seconds</li> <p> | ||
- | (This was repeated for another | + | (This was repeated for another 35 cycles) |
<li> 72˚C for 10 minutes </li> | <li> 72˚C for 10 minutes </li> | ||
<li> 4˚C to end. </li> <p> | <li> 4˚C to end. </li> <p> | ||
+ | <li type="disc"> The second PCR program was set up as per the following. There were two parts to this PCR to allow for the (AT-BHO-F) primer to anneal properly after initial annealing of the (AT-BHO-R) primer. </li> <p> | ||
- | + | <li>94˚C for 2 minutes </li> <p> | |
- | <li> | + | |
<li>94˚C for 30 seconds</li> | <li>94˚C for 30 seconds</li> | ||
<li> 40˚C for 30 seconds</li> | <li> 40˚C for 30 seconds</li> | ||
+ | <li>72˚C for 2 minutes and 30 seconds</li> <p> | ||
+ | |||
+ | <p>(This was repeated for another 4 cycles)<p> | ||
+ | |||
+ | <li type="disc"> Now we can add the (AT-BHO-F) primer to the reaction. </li> <p> | ||
+ | |||
+ | <li>4˚C for 5 minutes </li> <p> | ||
+ | |||
+ | <li>94˚C for 30 seconds</li> | ||
+ | <li> 60˚C for 30 seconds</li> | ||
<li>72˚C for 2 minutes and 30 seconds</li> | <li>72˚C for 2 minutes and 30 seconds</li> | ||
- | <p>(This was repeated for another 31 cycles) | + | <p>(This was repeated for another 31 cycles)<p> |
<li>72˚C for 10 minutes</li> | <li>72˚C for 10 minutes</li> | ||
- | <li> | + | <li> 4˚C to end </li> |
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <li type="disc">The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization </li> | ||
+ | |||
+ | |||
+ | |||
+ | <h4> Experimental Design – Primer combinations and annealing temperatures: | ||
+ | </h4> | ||
+ | |||
+ | <center> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th> Template </th> | ||
+ | <th> Primer pair </th> | ||
+ | <th> Annealing temp (degrees Celsius) </th> | ||
+ | <th> Number of cycles </th> | ||
+ | |||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> (AT-FWD-RBS)-(AT-RVS-1)PCR product </td> | ||
+ | <td> (AT-BHO-F)-(AT-BHO-R)</td> | ||
+ | <td> 40, then 60</td> | ||
+ | <td> 4, then 35 </td> | ||
+ | |||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> (AT-FWD-RBS)-(AT-AHO-R)PCR product </td> | ||
+ | <td> (AT-AHO-F)-(AT-AHO-R)</td> | ||
+ | <td> 60</td> | ||
+ | <td>35</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td> (AT-FWD-RBS)-(AT-RVS-1)PCR product (1:10 dilution) </td> | ||
+ | <td> (AT-BHO-F)-(AT-BHO-R)</td> | ||
+ | <td>40, then 60 </td> | ||
+ | <td> 4 then 35</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td> (AT-FWD-RBS)-(AT-AHO-R)PCR product (1:10 dilution)</td> | ||
+ | <td> (AT-AHO-F)-(AT-AHO-R)</td> | ||
+ | <td> 60 </td> | ||
+ | <td>35 </td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | <p> | ||
+ | |||
+ | |||
+ | <h3> <center> Figure 5. PCR optimization (with ssPCR amplification for (AT-BHO-F)primer) </h3></center></big> <p> | ||
+ | <center> | ||
+ | |||
+ | <a target='_blank' title='ImageShack - Image And Video Hosting' href='http://img840.imageshack.us/i/gel5.png/'><img src='http://img840.imageshack.us/img840/4507/gel5.png' border='0'/></a> | ||
+ | </a> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </center> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <b>Figure 5.</b> GelRed post-stained 2% agarose gel of optimized PCR using primer pairs for insertion of the heme oxygenase gene and bacteriophytochrome gene. In lanes 1 and 6 there is a 1kb ladder. In lane 2 there is the (AT-FWD-RBS)-(AT-RVS-1)PCR product amplified with (AT-BHO-F)-(AT-BHO-R)primer pair on the special PCR program allowing for the (AT-BHO-R)primer to bind first. In lane 3 there is the (AT-FWD-RBS)-(AT-AHO-R)PCR product amplified with (AT-AHO-F)-(AT-AHO-R)primer pair on the normal PCR program. In lane 4 there is the (AT-FWD-RBS)-(AT-RVS-1)PCR product diluted 1:10 amplified with (AT-BHO-F)-(AT-BHO-R)primer pair on the special PCR program allowing for the (AT-BHO-R)primer to bind first. In lane 5 there is the (AT-FWD-RBS)-(AT-AHO-R) PCR product diluted 1:10 amplified with (AT-AHO-F)-(AT-AHO-R)primer pair on the normal PCR program. <u> There is a product band seen in lane 3 – SUCCESS! This is the product with the bacteriophytochrome gene inserted AFTER the heme oxygenase gene. <p> | ||
+ | </u> | ||
+ | |||
+ | </p> | ||
+ | <p> | ||
+ | <b> Discussion of gradient PCR: </b> </p> <p> | ||
+ | |||
+ | The gradient PCR was used because the (AT-BHO-R) primer that was ordered was 12 base pairs shorter than what was supposed to be ordered. The (AT-BHO-R)primer was supposed to include the whole reverse primer sequence (18 base pairs) as well as an additional sequence for the HO site. This caused an issue with this primer annealing in previous PCR reactions. The gradient PCR will allow for this primer to anneal prior to any other primer annealing increasing the chance of annealing. </p> | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | |||
Latest revision as of 06:38, 27 October 2010
PROJECT LAB BOOK
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
- The first primer pair is (AT-BHO-F) with (AT-BHO-R) [this will insert the bacteriophytchrome gene BEFORE the heme oxygenase gene in the operon]
- The second primer pair is (AT-AHO-F) with (AT-AHO-R) [this will insert the bacteriophytochrome gene AFTER the heme oxygenase gene in the operon]
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 25 cycles)
Experimental Design – Primer combinations and annealing temperatures:
Fwd Primer | Rvs primer | Temp 1 (Degrees Celsius) | Temp 2 (Degrees Celsius) | Temp 3 (Degrees Celsius) | Temp 4 (Degrees Celsius) | Temp 5 (Degrees Celsius) |
---|---|---|---|---|---|---|
(AT-BHO-F) | (AT-BHO-R) | 57.1 | 58.7 | 60.6 | 63.4 | 64.8 |
(AT-AHO-F) | (AT-AHO-R) | 57.1 | 58.7 | 60.6 | 63.4 | 64.8 |
Figure 4. PCR Optimisation (gradient PCR) results
No products were observed. The anticipated product size was between 1.6kb and 3kb. The gel was over run but the only products that were over run were probably primer dimers, which are less than 300bp in size. = FAIL!
7th September 2010
PCR Optimization (using Gradient PCR)
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 25 cycles)
Experimental Design – Primer combinations and annealing temperatures:
DNA template | Dilution | Fwd primer | Rvs primer | Annealing temp (Degrees Celsius) |
---|---|---|---|---|
PCR product (from DNA2.2) | 1:100 | (AT-BHO-F) | (AT-BHO-R) | 60 |
PCR product (from DNA2.2) | 1:200 | (AT-BHO-F) | (AT-BHO-R) | 60 |
PCR product (from DNA2.2) | 1:100 | (AT-AHO-F) | (AT-AHO-R) | 60 |
PCR product (from DNA2.2) | 1:200 | (AT-AHO-F) | (AT-AHO-R) | 60 |
PCR product (from DNA2.2) | 1:100 | (AT-BHO-F) | (AT-BHO-R) | 65 |
PCR product (from DNA2.2) | 1:200 | (AT-BHO-F) | (AT-BHO-R) | 65 |
PCR product (from DNA2.2) | 1:100 | (AT-AHO-F) | (AT-AHO-R) | 65 |
PCR product (from DNA2.2) | 1:200 | (AT-AHO-F) | (AT-AHO-R) | 65 |
10th September 2010
PCR Optimization (repeated)
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The first PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 35 cycles)
The second PCR program was set up as per the following (this was for the amplification of product using the (AT-FWD-RBS) and (AT-AHO-R) primer pair):
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 40˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
- The second PCR program was set up as per the following. There were two parts to this PCR to allow for the (AT-BHO-F) primer to anneal properly after initial annealing of the (AT-BHO-R) primer.
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 40˚C for 30 seconds
- 72˚C for 2 minutes and 30 seconds
- Now we can add the (AT-BHO-F) primer to the reaction.
- 4˚C for 5 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes and 30 seconds
- 72˚C for 10 minutes
- 4˚C to end
- The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization
(This was repeated for another 35 cycles)
(This was repeated for another 4 cycles)
(This was repeated for another 31 cycles)
Experimental Design – Primer combinations and annealing temperatures:
Template | Primer pair | Annealing temp (degrees Celsius) | Number of cycles |
---|---|---|---|
(AT-FWD-RBS)-(AT-RVS-1)PCR product | (AT-BHO-F)-(AT-BHO-R) | 40, then 60 | 4, then 35 |
(AT-FWD-RBS)-(AT-AHO-R)PCR product | (AT-AHO-F)-(AT-AHO-R) | 60 | 35 |
(AT-FWD-RBS)-(AT-RVS-1)PCR product (1:10 dilution) | (AT-BHO-F)-(AT-BHO-R) | 40, then 60 | 4 then 35 |
(AT-FWD-RBS)-(AT-AHO-R)PCR product (1:10 dilution) | (AT-AHO-F)-(AT-AHO-R) | 60 | 35 |
Figure 5. PCR optimization (with ssPCR amplification for (AT-BHO-F)primer)
Discussion of gradient PCR:
The gradient PCR was used because the (AT-BHO-R) primer that was ordered was 12 base pairs shorter than what was supposed to be ordered. The (AT-BHO-R)primer was supposed to include the whole reverse primer sequence (18 base pairs) as well as an additional sequence for the HO site. This caused an issue with this primer annealing in previous PCR reactions. The gradient PCR will allow for this primer to anneal prior to any other primer annealing increasing the chance of annealing.