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/Glossary">Glossary</a></li> | <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> | ||
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<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><p> | ||
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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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<h4>Nanodrop absorbance readings: </h4> | <h4>Nanodrop absorbance readings: </h4> | ||
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<table> | <table> | ||
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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> | ||
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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> | ||
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<h4>Experimental Design – Primer combinations: </h4> | <h4>Experimental Design – Primer combinations: </h4> | ||
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- | A band was seen in lane 9. This lane was using the primer and DNA template combinations of DNA2.2, | + | 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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<hr> | <hr> | ||
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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> | ||
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<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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<h4>Experimental Design – Primer combinations: </h4> | <h4>Experimental Design – Primer combinations: </h4> | ||
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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> |
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<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> | ||
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<h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | <h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | ||
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7th September 2010 <p> | 7th September 2010 <p> | ||
- | PCR Optimization (using Gradient PCR) <p> </big> </hr> <p> </font></ | + | PCR Optimization (using Gradient PCR) <p> </big> </hr> <p> </font></b> |
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<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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<h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | <h4>Experimental Design – Primer combinations and annealing temperatures: </h4> | ||
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10th September 2010 <p> | 10th September 2010 <p> | ||
- | PCR Optimization (repeated)<p> </big> </hr> <p> </font></ | + | PCR Optimization (repeated)<p> </big> </hr> <p> </font></b> |
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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.