Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminB2"
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We performed again the colony PCR (with o15.097 and o15.098) on the same colonies with a different DreamTaq enzyme. | We performed again the colony PCR (with o15.097 and o15.098) on the same colonies with a different DreamTaq enzyme. | ||
− | PCR analysis on 1% agarose 0.5X TAE gel.<br> | + | PCR analysis on 1% agarose 0.5X TAE gel.<br><br> |
− | <br> | + | |
<img width="20%" src="https://static.igem.org/mediawiki/2015/f/f0/PB_notebookB2_pic3.jpg"/> | <img width="20%" src="https://static.igem.org/mediawiki/2015/f/f0/PB_notebookB2_pic3.jpg"/> | ||
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So, we PCRed two of our gBlocks (RibE and T25) with the three different DreamTaqs (Extension time: 2.00min).<br> | So, we PCRed two of our gBlocks (RibE and T25) with the three different DreamTaqs (Extension time: 2.00min).<br> | ||
Then, to see whether the amplification worked of not, we run our PCR products on an 1% agarose gel.<br> | Then, to see whether the amplification worked of not, we run our PCR products on an 1% agarose gel.<br> | ||
− | Here is a picture of the gel after 20min at 100V:<br> | + | Here is a picture of the gel after 20min at 100V:<br><br> |
+ | |||
− | |||
<img width="20%" src="https://static.igem.org/mediawiki/2015/f/f1/PB_notebookB2_pic11.jpg"/><br> | <img width="20%" src="https://static.igem.org/mediawiki/2015/f/f1/PB_notebookB2_pic11.jpg"/><br> | ||
<b>Figure 5:</b><i> PCR amplification using three DreamTaq mixes unraveled by electrophoresis</i> | <b>Figure 5:</b><i> PCR amplification using three DreamTaq mixes unraveled by electrophoresis</i> | ||
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Because of the poor digestion capacity we observed in BbsI, we investigated on the net if some other people had problems with the NEB BbsI enzyme. [This website] explained that they detected that "BbsI supplied by NEB (an isoschizomer of BpiI) loses activity extremely quickly and has a lot of star-activity". Using this information, we thought about using BpiI instead of BbsI.<br> | Because of the poor digestion capacity we observed in BbsI, we investigated on the net if some other people had problems with the NEB BbsI enzyme. [This website] explained that they detected that "BbsI supplied by NEB (an isoschizomer of BpiI) loses activity extremely quickly and has a lot of star-activity". Using this information, we thought about using BpiI instead of BbsI.<br> | ||
To confirm that BpiI would digest our plasmid the same way than BbsI, we digested p15.01 with BpiI to see if it would produce the same three bands than BbsI.<br> | To confirm that BpiI would digest our plasmid the same way than BbsI, we digested p15.01 with BpiI to see if it would produce the same three bands than BbsI.<br> | ||
− | Figure 6 shows the electrophoresis result of p15.01 after digestion with Eco31I and BpiI:<br> | + | Figure 6 shows the electrophoresis result of p15.01 after digestion with Eco31I and BpiI:<br><br> |
<img width="10%" src="https://static.igem.org/mediawiki/2015/a/af/PB_notebookB2_pic14.jpg"/><br> | <img width="10%" src="https://static.igem.org/mediawiki/2015/a/af/PB_notebookB2_pic14.jpg"/><br> | ||
<b>Figure 6:</b> <i> Digestion of p15.01 with BpiI</i><br> | <b>Figure 6:</b> <i> Digestion of p15.01 with BpiI</i><br> | ||
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Miniprep of the 14 overnight cultures and restriction analysis of their plasmid with BpiI and Eco31I.<br> | Miniprep of the 14 overnight cultures and restriction analysis of their plasmid with BpiI and Eco31I.<br> | ||
− | Here is a simulation of an electrophoresis:<br> | + | Here is a simulation of an electrophoresis:<br><br> |
<img width="20%" src="https://static.igem.org/mediawiki/2015/f/f3/PB_notebookB2_gel_simu2.png"/><br> | <img width="20%" src="https://static.igem.org/mediawiki/2015/f/f3/PB_notebookB2_gel_simu2.png"/><br> | ||
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<br> | <br> | ||
− | Restriction analysis result after 25 min (100V, 1% agarose TAE gel):<br> | + | Restriction analysis result after 25 min (100V, 1% agarose TAE gel):<br><br> |
<img width="40%" src="https://static.igem.org/mediawiki/2015/a/a1/PB_notebookB2_pic13.jpg"/><br> | <img width="40%" src="https://static.igem.org/mediawiki/2015/a/a1/PB_notebookB2_pic13.jpg"/><br> | ||
<b>Figure 8:</b> <i> Electrophoresis of the restriction analysis of transformant's plasmid with BpiI and Eco31I </i> <br> | <b>Figure 8:</b> <i> Electrophoresis of the restriction analysis of transformant's plasmid with BpiI and Eco31I </i> <br> | ||
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To check whether the assembly is working or not, we ran a gel to see if the Golden Gate is producing some expected bands.<br> | To check whether the assembly is working or not, we ran a gel to see if the Golden Gate is producing some expected bands.<br> | ||
− | Here is a picture after 20 minutes of migration:<br> | + | Here is a picture after 20 minutes of migration:<br><br> |
<img width="20%" src="https://static.igem.org/mediawiki/2015/d/d3/PB_notebookB2_pic15.jpg"/><br> | <img width="20%" src="https://static.igem.org/mediawiki/2015/d/d3/PB_notebookB2_pic15.jpg"/><br> | ||
<b>Figure 9: </b><i>Electrophoresis of p15.01, RibA, D, E and T after Golden gate assembly</i><br> | <b>Figure 9: </b><i>Electrophoresis of p15.01, RibA, D, E and T after Golden gate assembly</i><br> | ||
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Because of a human mistake, thermocycler lid was inversed, letting the caps opening themselves when temperature increased. The PCR products were lost.<br><br> | Because of a human mistake, thermocycler lid was inversed, letting the caps opening themselves when temperature increased. The PCR products were lost.<br><br> | ||
− | Restriction digestion of p15.01 by BbsI or BpiI(Fast Digest), then by Eco31I.<br> | + | Restriction digestion of p15.01 by BbsI or BpiI(Fast Digest), then by Eco31I.<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/7/77/PB_notebookB2_pic18.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/7/77/PB_notebookB2_pic18.jpg"/><br> | ||
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Second PCR amplification of Rib gblocks. Final volume was 100µL, elongation time 2.30 minutes and annealing temperature was 64°C.<br> | Second PCR amplification of Rib gblocks. Final volume was 100µL, elongation time 2.30 minutes and annealing temperature was 64°C.<br> | ||
− | Electrophoresis was performed to control the gBlocks amplification.<br> | + | Electrophoresis was performed to control the gBlocks amplification.<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/4/47/PB_notebookB2_pic19.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/4/47/PB_notebookB2_pic19.jpg"/><br> | ||
<b>Figure 12: </b><i>Rib gblocks amplification (64°C annealing) control by electrophoresis</i><br> | <b>Figure 12: </b><i>Rib gblocks amplification (64°C annealing) control by electrophoresis</i><br> | ||
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− | It clearly appeared that something was wrong the amplification. Because of the absence of band for RibA and the double band for RibD, we decided to perform a third PCR using a lower annealing temperature (55°C).<br> | + | It clearly appeared that something was wrong the amplification. Because of the absence of band for RibA and the double band for RibD, we decided to perform a third PCR using a lower annealing temperature (55°C).<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/7/72/PB_notebookB2_pic20.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/7/72/PB_notebookB2_pic20.jpg"/><br> | ||
<b>Figure 13: </b><i>Rib gblocks amplification (55°C annealing) control by electrophoresis</i><br> | <b>Figure 13: </b><i>Rib gblocks amplification (55°C annealing) control by electrophoresis</i><br> | ||
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generuler 1kb - RibA - RibD - RibE - RibT25 - RibT48</i><br><br> | generuler 1kb - RibA - RibD - RibE - RibT25 - RibT48</i><br><br> | ||
All the bands correspond to the expected size of the gBlocks, but RibA for which no band was visible.<br><br> | All the bands correspond to the expected size of the gBlocks, but RibA for which no band was visible.<br><br> | ||
− | Thus, we decided to perform a gradient PCR (five tubes spread from 52°C to 64°C) on RibA to determine what was the best (or a least a good) temperature for annealing.<br> | + | Thus, we decided to perform a gradient PCR (five tubes spread from 52°C to 64°C) on RibA to determine what was the best (or a least a good) temperature for annealing.<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/7/74/PB_notebookB2_pic22.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/7/74/PB_notebookB2_pic22.jpg"/><br> | ||
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<br><br><h1 class="date">14/08</h1><br><br> | <br><br><h1 class="date">14/08</h1><br><br> | ||
− | Colony PCR of 15 colonies from each transformation. Annealing temperature was 64°C and elongation set to 3 minutes.<br> | + | Colony PCR of 15 colonies from each transformation. Annealing temperature was 64°C and elongation set to 3 minutes.<br><br> |
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Even with such an ugly gel (a very old stock of agarose was used), one can clearly see that four of the five gBlocks are correctly amplified. RibE expected size is 0.9kb, here an 1.5 and a 0.5 bands are visible.<br><br> | Even with such an ugly gel (a very old stock of agarose was used), one can clearly see that four of the five gBlocks are correctly amplified. RibE expected size is 0.9kb, here an 1.5 and a 0.5 bands are visible.<br><br> | ||
− | To determine the optimal Tm for RibE amplification, we performed a gradient PCR from 55°C to 64°C.<br> | + | To determine the optimal Tm for RibE amplification, we performed a gradient PCR from 55°C to 64°C.<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/5/50/PB_notebookB2_pic27.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/5/50/PB_notebookB2_pic27.jpg"/><br> | ||
<b>Figure 21: </b><i>RibE gradient PCR amplification</i><br> | <b>Figure 21: </b><i>RibE gradient PCR amplification</i><br> | ||
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Miniprep of the four overnight cultures and electrophoresis of the extracted DNA.<br> | Miniprep of the four overnight cultures and electrophoresis of the extracted DNA.<br> | ||
− | Restriction analysis of the plasmid using BpiI and Eco31I.<br> | + | Restriction analysis of the plasmid using BpiI and Eco31I.<br><br> |
<img width="30%" src="https://static.igem.org/mediawiki/2015/9/97/PB_notebookB2_pic29.jpg"/><br> | <img width="30%" src="https://static.igem.org/mediawiki/2015/9/97/PB_notebookB2_pic29.jpg"/><br> | ||
<b>Figure 22: </b><i>Restriction analysis of p15.06 transformants</i><br> | <b>Figure 22: </b><i>Restriction analysis of p15.06 transformants</i><br> | ||
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The digestion profile is showing no difference with the negative control. It meant that none of these three transformants contained the inserts.<br><br> | The digestion profile is showing no difference with the negative control. It meant that none of these three transformants contained the inserts.<br><br> | ||
− | PCR amplification of RibD (o15.003+o15.004), RibE (o15.005+o15.006) and both ribT25/48(o15.007+o15.008/o15.009+o15.010).<br> | + | PCR amplification of RibD (o15.003+o15.004), RibE (o15.005+o15.006) and both ribT25/48(o15.007+o15.008/o15.009+o15.010).<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/5/5d/PB_notebookB2_pic30.jpg"/> | <img width="15%" src="https://static.igem.org/mediawiki/2015/5/5d/PB_notebookB2_pic30.jpg"/> | ||
<img width="15%" src="https://static.igem.org/mediawiki/2015/2/22/PB_notebookB2_pic31.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/2/22/PB_notebookB2_pic31.jpg"/><br> | ||
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RibD was presenting a second amplification band and RibE, T25 and T48 were all not visible.<br> | RibD was presenting a second amplification band and RibE, T25 and T48 were all not visible.<br> | ||
− | We launched an other PCR for RibD and RibE using a different phusion master mix.<br> | + | We launched an other PCR for RibD and RibE using a different phusion master mix.<br><br> |
<img width="30%" src="https://static.igem.org/mediawiki/2015/5/59/PB_notebookB2_pic32.jpg"/><br> | <img width="30%" src="https://static.igem.org/mediawiki/2015/5/59/PB_notebookB2_pic32.jpg"/><br> | ||
<b>Figure 24: </b><i>Electrophoresis of digested an undigested p15.01(BpiI) and RibD and E amplification control</i><br> | <b>Figure 24: </b><i>Electrophoresis of digested an undigested p15.01(BpiI) and RibD and E amplification control</i><br> | ||
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<br><br><h1 class="date">16/08</h1><br><br> | <br><br><h1 class="date">16/08</h1><br><br> | ||
− | 15/08 gBlocks PCR amplification analysis by electrophoresis.<br> | + | 15/08 gBlocks PCR amplification analysis by electrophoresis.<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/a/a6/PB_notebookB2_pic36.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/a/a6/PB_notebookB2_pic36.jpg"/><br> | ||
<b>Figure 25: </b><i>gBlocks PCR amplification control by Electrophoresis</i><br> | <b>Figure 25: </b><i>gBlocks PCR amplification control by Electrophoresis</i><br> | ||
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<br><br><h1 class="date">17/08</h1><br><br> | <br><br><h1 class="date">17/08</h1><br><br> | ||
− | Electrophoresis of RibD gradient PCR.<br> | + | Electrophoresis of RibD gradient PCR.<br><br> |
<img width="15%" src="https://static.igem.org/mediawiki/2015/0/03/PB_notebookB2_pic33.jpg"/><br> | <img width="15%" src="https://static.igem.org/mediawiki/2015/0/03/PB_notebookB2_pic33.jpg"/><br> | ||
<b>Figure 26: </b><i>Electrophoresis of RibD amplification with eight annealing temperature</i><br> | <b>Figure 26: </b><i>Electrophoresis of RibD amplification with eight annealing temperature</i><br> | ||
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PCR amplification of RibD, RibE, RibT25 and RibT48 (two mix for each, 1µL of dNTPS was added to increase the yield, 50µL of mix per tube).<br> | PCR amplification of RibD, RibE, RibT25 and RibT48 (two mix for each, 1µL of dNTPS was added to increase the yield, 50µL of mix per tube).<br> | ||
− | For annealing , we used respectively 60.5, 55, 64 and 62.2°C for RibD, RibE, RibT25 and RibT48.<br> | + | For annealing , we used respectively 60.5, 55, 64 and 62.2°C for RibD, RibE, RibT25 and RibT48.<br><br> |
<img width="25%" src="https://static.igem.org/mediawiki/2015/d/d6/PB_notebookB2_pic37.jpg"/><br> | <img width="25%" src="https://static.igem.org/mediawiki/2015/d/d6/PB_notebookB2_pic37.jpg"/><br> | ||
<b>Figure 28: </b><i>Electrophoresis of Rib gBlocks PCR products</i><br> | <b>Figure 28: </b><i>Electrophoresis of Rib gBlocks PCR products</i><br> | ||
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<br><br><h1 class="date">19/08</h1><br><br> | <br><br><h1 class="date">19/08</h1><br><br> | ||
− | RibD gradient PCR amplification was controled by electrophoresis.<br> | + | RibD gradient PCR amplification was controled by electrophoresis.<br><br> |
<img width="25%" src="https://static.igem.org/mediawiki/2015/6/69/PB_notebookB2_pic38.jpg"/><br> | <img width="25%" src="https://static.igem.org/mediawiki/2015/6/69/PB_notebookB2_pic38.jpg"/><br> | ||
<b>Figure 29: </b><i>Control of RibD gradient PCR amplification by Electrophoresis</i><br> | <b>Figure 29: </b><i>Control of RibD gradient PCR amplification by Electrophoresis</i><br> | ||
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PCR purification of RibE, RibT25 and ribT48 (amplified the 18/08).<br> | PCR purification of RibE, RibT25 and ribT48 (amplified the 18/08).<br> | ||
− | Purification products were run on a 1% agarose gel to control their purity.<br> | + | Purification products were run on a 1% agarose gel to control their purity.<br><br> |
<img width="25%" src="https://static.igem.org/mediawiki/2015/f/f7/PB_notebookB2_pic39.jpg"/><br> | <img width="25%" src="https://static.igem.org/mediawiki/2015/f/f7/PB_notebookB2_pic39.jpg"/><br> | ||
<b>Figure 30: </b><i>Electrophoresis of amplified and PCR purified RibE, RibT25 and Ribt48</i><br> | <b>Figure 30: </b><i>Electrophoresis of amplified and PCR purified RibE, RibT25 and Ribt48</i><br> |
Revision as of 14:37, 24 August 2015