Team:Marburg:Project:Notebook:October
From 2014.igem.org
(Difference between revisions)
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- | <p>After the purification steps until the ammonium | + | <p>After the purification steps until the ammonium sulphate precipitation a SDS-gel with coomassie staining was done and WT3610 as well as Hag-D2_3 were used as a control. </p> |
<img src="https://static.igem.org/mediawiki/2014/2/2c/MR_2014-10-01_18.72.jpg" width="30%" /> | <img src="https://static.igem.org/mediawiki/2014/2/2c/MR_2014-10-01_18.72.jpg" width="30%" /> | ||
- | <p>The gel shows that there is a | + | <p>The gel shows that there is a band after the ammonium sulfate precipitation which is running to low to be flagellin. The isolation was repeated several times with the same result so that we think of unstable flagellins. Additionally, a <i>B. subtilis</i> PY79 strain with a Strep-Tag integrated into the normal Hag-gene was used for isolation of flagella. They might be more stable than Hag-D2-Strep filaments.</p> |
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<legend><a name="exp23.35">23.35 Mutagenesis PCR of pSB1C3 Hag-D2-Strep clone 1 and 2</a></legend> | <legend><a name="exp23.35">23.35 Mutagenesis PCR of pSB1C3 Hag-D2-Strep clone 1 and 2</a></legend> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>Mutagenesis Primer arrived → PCR with | + | <p>Mutagenesis Primer arrived → PCR with pSB1C3-Hag-<i>Kpn</i>I-Strep clone 1 and 2 (appr. 20 ng/µl) in 30 µl reaction</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
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- | <th scope="row">Phusion</th> | + | <th scope="row">Phusion DNA-polymerase</th> |
<td>1</td> | <td>1</td> | ||
</tr> | </tr> | ||
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<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The structure of the flagella of the mutant strains <em>Bacillus subtilis</em> 3610 D2-Strep and <em>Bacillus subtilis</em> 3610 D2-cup should be investigated by electron microscopy. Thus cultures of strain WT3619, 3610 D2-Strep and 3610 D2-Cup were inoculated in 5 ml LB medium. The cultures were incubated shaking at 37°C until they reached the exponential phase with an | + | <p>The structure of the flagella of the mutant strains <em>Bacillus subtilis</em> 3610 D2-Strep and <em>Bacillus subtilis</em> 3610 D2-cup should be investigated by electron microscopy. Thus cultures of strain WT3619, 3610 D2-Strep and 3610 D2-Cup were inoculated in 5 ml LB medium. The cultures were incubated shaking at 37°C until they reached the exponential phase with an OD<sub>600</sub> of 0.7 - 0.8, and until the cells reached the stationary phase. The cells were kept on the same OD by storage on ice until further processing for electron microscopy.</p> |
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- | <p>The purified Hag-D2-Strep which was left from crystallization and | + | <p>The purified Hag-D2-Strep which was left from crystallization and the purified Hag-Strep were used to make a pulldown with Streptavidin beads. We wanted to see if the Strep-Tag in the flagellin monomers can interact with the Streptavidin beads.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
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<ul class="list"> | <ul class="list"> | ||
<li>Pd-column + bead-slurry/ 1 min. 4000 rpm, RT</li> | <li>Pd-column + bead-slurry/ 1 min. 4000 rpm, RT</li> | ||
- | <li>Add 400 | + | <li>Add 400 µL GeFi(old) - wash/ 1 min. 4000 rpm, RT</li> |
- | <li>Discard Flow through, add 400 | + | <li>Discard Flow through, add 400 µL GeFi(old) + Protein → 20 min on Turning wheel at RT</li> |
<li>1 min at 4000 rpm, RT</li> | <li>1 min at 4000 rpm, RT</li> | ||
- | <li>2 x 400 µL | + | <li>2 x 400 µL "Strep-wash" </li> |
- | <li>Elution with 40 µL | + | <li>Elution with 40 µL 'Strep-Elution'</li> |
<li>5 min. incubation at RT</li> | <li>5 min. incubation at RT</li> | ||
<li>1 min. 4000 rpm at RT, Elution in 10 µL Loading Buffer in 1,5 mL Eppi</li> | <li>1 min. 4000 rpm at RT, Elution in 10 µL Loading Buffer in 1,5 mL Eppi</li> | ||
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<div class="exp-content"> | <div class="exp-content"> | ||
<p>Inoculate 16 clones from the Gibson assembly (1.10.) in Lb-Cm for plasmid preparation and test restriction.</p> | <p>Inoculate 16 clones from the Gibson assembly (1.10.) in Lb-Cm for plasmid preparation and test restriction.</p> | ||
- | <p>Small colonies on the Gibson transformation plates are visible in the morning; further incubation until colonies can be inoculated in LB-Cm. Plasmids will then be isolated from the cultures and digested with | + | <p>Small colonies on the Gibson transformation plates are visible in the morning; further incubation until colonies can be inoculated in LB-Cm. Plasmids will then be isolated from the cultures and digested with <i>Pst</i>I in order to analyze the removal of the <i>Pst</i>I restriction site in the insert.</p> |
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<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>In order to fuse the genes for streptavidin and cup, which both had overhangs for each other, a Gibson assembly was carried out. Two ratios of DNA addition were tested: equal volumes of streptavidin and cup were added to the Gibson mix and the other ratio was calculated according to the formula in 13.102 with R = 1. A third Gibson reaction was prepared with the digested vector pET16b, since it was thought, the primers for strep-cup were designed with overhangs to this vector. Although the vector maps show only the overhangs with restriction sites for | + | <p>In order to fuse the genes for streptavidin and cup, which both had overhangs for each other, a Gibson assembly was carried out. Two ratios of DNA addition were tested: equal volumes of streptavidin and cup were added to the Gibson mix and the other ratio was calculated according to the formula in 13.102 with R = 1. A third Gibson reaction was prepared with the digested vector pET16b, since it was thought, the primers for strep-cup were designed with overhangs to this vector. Although the vector maps show only the overhangs with restriction sites for <i>Nco</i>I and <i>Sac</i>I the reaction was prepared with the vector as well.</p> |
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<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>In order to check the interaction of StrepDARPidin with the EpCAM on A549 Lung Carcinoma cells we planned an ELISA-Like assay again. Yesterday row F 1-12 of a black Fluotrac600 96-well plate was coated with 100000 cells A549/ well (counted with Neubauer-Chamber) overnight at | + | <p>In order to check the interaction of StrepDARPidin with the EpCAM on A549 Lung Carcinoma cells we planned an ELISA-Like assay again. Yesterday row F 1-12 of a black Fluotrac600 96-well plate was coated with 100000 cells A549/ well (counted with Neubauer-Chamber) overnight at 37°C. 100000 cells of 3T3 fibroblasts were used as negative control and as well used to coat well G6.</p> |
<p>We intended to incubate them with different concentrations of StrepDARPidin and then target the N-terminal His-Tag with and Anti-His antibody Alexa488 conjugated (1:50 endvolumen). </p> | <p>We intended to incubate them with different concentrations of StrepDARPidin and then target the N-terminal His-Tag with and Anti-His antibody Alexa488 conjugated (1:50 endvolumen). </p> | ||
<p>In wells H1-6 gel filtration purified StrepDARPidin with 87 µM was tested, in well H 7-12 Ni-NTA purified StrepDARPidin with 280 µM.</p> | <p>In wells H1-6 gel filtration purified StrepDARPidin with 87 µM was tested, in well H 7-12 Ni-NTA purified StrepDARPidin with 280 µM.</p> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The purified Hag-D2-Strep which was left from crystallization and | + | <p>The purified Hag-D2-Strep which was left from crystallization and the purified Hag-Strep were used to make a pulldown with purified StrepDARPidin. We wanted to see if the Strep-Tag in the flagellin monomers can interact with the StrepDARPidin, especially with the Streptavidin part.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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<legend><a name="exp23.40">23.40 Plasmid isolation and test restriction of Hag-D2-Strep clone 1 and 2 (mutagenesis)</a></legend> | <legend><a name="exp23.40">23.40 Plasmid isolation and test restriction of Hag-D2-Strep clone 1 and 2 (mutagenesis)</a></legend> | ||
<div class="aim"> | <div class="aim"> | ||
- | <p>Aim: Analyze the removal of the | + | <p>Aim: Analyze the removal of the <i>Pst</i>I restriction site </p> |
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>Plasmid preparation of the inoculated E. coli cultures was performed after over night incubation. 500-600 ng of all plasmids have been digested with | + | <p>Plasmid preparation of the inoculated E. coli cultures was performed after over night incubation. 500-600 ng of all plasmids have been digested with <i>Pst</i>I for 2h (37°C). In case the <i>Pst</i>I restriction site is removed from the insert, only one fragment (linearized plasmid) should occur in the gel. If the mutagenesis PCR failed and the restriction site is still inside the plasmid 2 fragments (3000bp + 470 bp) should occur in the gel.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <th scope="row"> | + | <th scope="row"><i>Pst</i>I</th> |
<td>0,5</td> | <td>0,5</td> | ||
<td>9</td> | <td>9</td> | ||
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<img src="https://static.igem.org/mediawiki/2014/6/62/MR_2014-10-03_23.40.png" width="40%" /> | <img src="https://static.igem.org/mediawiki/2014/6/62/MR_2014-10-03_23.40.png" width="40%" /> | ||
<p>Negative clones: 1.2, 1.4, 2.3 and 2.9</p> | <p>Negative clones: 1.2, 1.4, 2.3 and 2.9</p> | ||
- | <p> | + | <p>Only one fragment should occur in the gel in case of <i>Pst</i>I linearization.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>Since the last ligation yielded in barely any colonies on the plates, the vectors were not further purified after restriction with | + | <p>Since the last ligation yielded in barely any colonies on the plates, the vectors were not further purified after restriction with <i>Nco</i>I and <i>Sac</i>I, which would result in a high loss of DNA. The ligation of piGEM007, piGEM008, piGEM009 with the IPTG inducible and the strong constitutive promoter and piGEM002 with the strong constitutive promoter was carried out. The DNA ratios were calculated according to the previously used formula (13.102).</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <th scope="row"> | + | <th scope="row"><i>Nco</i>I</th> |
<td>0,5</td> | <td>0,5</td> | ||
</tr> | </tr> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>Colony PCRs were performed to screen the clones for the right plasmids. Every plate carried enough colonies to pick multiple clones. Five clones of every plate was picked and this time instead of using the common gfp-reverse primer iGEM006 the primers specific for the degradation tags of the different nose plasmids were used. The ligation with the constitutive promoter should be repeated, since the fragment was not digested with | + | <p>Colony PCRs were performed to screen the clones for the right plasmids. Every plate carried enough colonies to pick multiple clones. Five clones of every plate was picked and this time instead of using the common gfp-reverse primer iGEM006 the primers specific for the degradation tags of the different nose plasmids were used. The ligation with the constitutive promoter should be repeated, since the fragment was not digested with <i>Nco</i>I and <i>Sac</i>I before.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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<legend><a name="exp23.41">23.41 Repeat test restriction</a></legend> | <legend><a name="exp23.41">23.41 Repeat test restriction</a></legend> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>New test restriction with only 200 ng plasmid DNA since the last restriction showed a lot of undigested DNA. In order to test for the removal of the | + | <p>New test restriction with only 200 ng plasmid DNA since the last restriction showed a lot of undigested DNA. In order to test for the removal of the <i>Pst</i>I restriction site 8 clones will be digested with <i>Pst</i>I and <i>Pst</i>I/<i>Eco</i>RI.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <th scope="row"> | + | <th scope="row"><i>Pst</i>I</th> |
<td>0,5</td> | <td>0,5</td> | ||
<td>4,5</td> | <td>4,5</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <th scope="row"> | + | <th scope="row">i>Eco</i>RI</th> |
<td>0,5</td> | <td>0,5</td> | ||
<td>4,5</td> | <td>4,5</td> | ||
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<br /> | <br /> | ||
<img src="https://static.igem.org/mediawiki/2014/9/9e/MR_2014-10-05_23.41.png" width="50%" /> | <img src="https://static.igem.org/mediawiki/2014/9/9e/MR_2014-10-05_23.41.png" width="50%" /> | ||
- | <p> | + | <p><i>Pst</i>I restriction should lead to a linearized plasmid, i>Eco</i>RI/<i>Pst</i>I shows the expected size of 1500 and 2000 bp. Additionally some undigested plasmid is left in the <i>Pst</i>I restriction.</p> |
<ul> | <ul> | ||
- | <li>Contamination of plasmid? New | + | <li>Contamination of plasmid? New digestion with new batch of <i>Pst</i>I and <i>Eco</i>RI.</li> |
- | <li> | + | <li><i>Pst</i>I restriction lead to a linearized plasmid and <i>Pst</i>I/<i>Eco</i>RI double digest lead to two fragments of a size of 1400 and 2000 bp</li> |
<li>Due to limited time clones 1.1 and 2.1 will be sent for sequencin</li> | <li>Due to limited time clones 1.1 and 2.1 will be sent for sequencin</li> | ||
</ul> | </ul> | ||
<img src="https://static.igem.org/mediawiki/2014/b/b8/MR_2014-10-05_23.41_2.png" width="50%" /> | <img src="https://static.igem.org/mediawiki/2014/b/b8/MR_2014-10-05_23.41_2.png" width="50%" /> | ||
- | <p>Result: | + | <p>Result: <i>Pst</i>I is contaminated with i>Eco</i>RI.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>Clone 6 was picked to inoculate a miniprep in LB-ampicillin. The plasmid was | + | <p>Clone 6 was picked to inoculate a miniprep in LB-ampicillin. The plasmid was isolated after 8 hours of incubation at 37°C. A control restriction was carried out with the enzymes <i>Nco</i>I, <i>Eco</i>RI and <i>Pst</i>I; a negative clone would result in bands of the size of 394 bp, 748 bp and 4569 bp; a positive clone would yield bands in size of 748 bp, 865 bp and 4569 bp. Lane 2 showed the fragments in the expected size.</p> |
</div> | </div> | ||
<img src="https://static.igem.org/mediawiki/2014/5/56/PET16b_StrepCup_restriction.png" width="20%" /> | <img src="https://static.igem.org/mediawiki/2014/5/56/PET16b_StrepCup_restriction.png" width="20%" /> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>To transform Bacillus a huge amount of plasmid is needed. For this reason 100 ml culture with <em>E. coli</em> for every nose plasmid with a metallosensitive promoter was harvested and the plasmids were | + | <p>To transform Bacillus a huge amount of plasmid is needed. For this reason 100 ml culture with <em>E. coli</em> for every nose plasmid with a metallosensitive promoter was harvested and the plasmids were isolated according to the protocol in the Qiagen Plasmid Plus Maxi Kit. One exception was made, since no QIAvac plus 24 was available and no vacuum pump. Thus the filtered cell lysate was loaded onto the columns by centrifugation. </p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>30 ml LB-ampicillin were inoculated with cells of <i>E. coli</i> BL21(DE3) pET16b_StrepCup from plate. This culture was incubated shaking at 37°C until they reached an | + | <p>30 ml LB-ampicillin were inoculated with cells of <i>E. coli</i> BL21(DE3) pET16b_StrepCup from plate. This culture was incubated shaking at 37°C until they reached an OD<sub>600</sub> of 0.58. A preinduction sample was taken (1.2 ml = (0.7 * 0.7) / (0.7 * 0.58)), the cells were pelleted by centrifugation at 14000 rpm for 1 minute, followed by resuspension in 80 µl water and 20 µl 5X SDS loading dye. The culture was induced with 30 µl IPTG for 3 hours. Another sample was taken afterwards, at this time point the culture reached an OD<sub>600</sub> of 1.2 (0.583 ml = (0.7 * 0.7 / 0.7 * 1.2)). These samples were boiled at 95°C for 10 minutes and then analyzed by SDS-PAGE (12% SDS-polyacrylamide gel). |
<br /> | <br /> | ||
<img src="https://static.igem.org/mediawiki/2014/f/f7/SDS.png" width="30%" /> | <img src="https://static.igem.org/mediawiki/2014/f/f7/SDS.png" width="30%" /> | ||
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<img src="https://static.igem.org/mediawiki/2014/4/42/CPCR_lac_plasmids_09.10.2014.png" width="30%" /> | <img src="https://static.igem.org/mediawiki/2014/4/42/CPCR_lac_plasmids_09.10.2014.png" width="30%" /> | ||
<br /> | <br /> | ||
- | <p>For the colony PCR several positive clones could be seen that exposed a band in the height of ca 1000 bp, clone 9 was chosen to work with in the further steps. The control PCR on the nose - lac plasmids was negative for all plasmids. Some showed two very thin bands of ca 750 bp and 1000 bp size. The expected fragment should have a size of | + | <p>For the colony PCR several positive clones could be seen that exposed a band in the height of ca 1000 bp, clone 9 was chosen to work with in the further steps. The control PCR on the nose - lac plasmids was negative for all plasmids. Some showed two very thin bands of ca 750 bp and 1000 bp size. The expected fragment should have a size of approx. 818 bp. These findings were striking, since the colony PCR for the clones from which the plasmids were isolated were positive.</p> |
<p>The control PCR for the plasmids piGEM002/007 + const and piGEM008 + Cu were positive. The clones containing these plasmids were used to inoculate 100 ml LB+ampicillin for a maxi prep.</p> | <p>The control PCR for the plasmids piGEM002/007 + const and piGEM008 + Cu were positive. The clones containing these plasmids were used to inoculate 100 ml LB+ampicillin for a maxi prep.</p> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The colony PCR for the clones containing the nose plasmids were positive but the control PCR with the | + | <p>The colony PCR for the clones containing the nose plasmids were positive but the control PCR with the isolated plasmids were negative. Since colony PCRs are a less reliable method than the control PCR on the isolated plasmid or a control restriction, the plasmids should be negative. To be sure a control restriction with <i>Bam</i>HI and <i>Hind</i>III was carried out with these plasmids. The positive plasmids should result in fragments of 4194 bp and 2620 bp size, the negative plasmid should not be digested by <i>Hind</I>III, because the only restriction site for <i>Hind</I>III is in the IPTG inducible promoter. As a control piGEM030 was cut with <i>Hind</I>III and <i>Nco</i>I (1172 bp, 5246 bp and 241 bp) and piGEM008 + const was cut with <i>Bam</i>HI and <i>Nco</i>I (4189 bp + 2757 for a positive plasmid, 4189 bp and 2518 bp for a negative plasmid). |
<br /> | <br /> | ||
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
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<img src="https://static.igem.org/mediawiki/2014/9/95/Kontroll_verdau_und_Colony_PCR_lac_plasmide_09.10.2014.png" width="40%" /> | <img src="https://static.igem.org/mediawiki/2014/9/95/Kontroll_verdau_und_Colony_PCR_lac_plasmide_09.10.2014.png" width="40%" /> | ||
<br /> | <br /> | ||
- | <p>The control restriction of the nose plasmids was negative, the plasmid just seemed to be linearized and not cut into two fragments. That means that the | + | <p>The control restriction of the nose plasmids was negative, the plasmid just seemed to be linearized and not cut into two fragments. That means that the <i>Hind</i>III could not cut since the IPTG inducible promoter was not contained in the plasmids. However, also the control piGEM030 did not show the expected bands. It could be possible that <i>Hind</i>III did not work correctly, since it was no HF enzyme and used in the CutSmart buffer of NEB, but according to the NEB Double Digest Finder <i>Hind</i>III was able to cut in CutSmart only with the half of its optimal activity. That was the reason, why the double amount of <I>Hind</i>III was chosen.</p> |
<p>Some samples of the new colony PCR showed bands slightly higher than 750 bp, which would resemble the size of the expected fragment of ca 800 bp, but since the controls with the nose plasmids containing the Ag sensitive promoter (all confirmed positive by control PCR on isolated plasmids) were not all positive, it was uncertain, that the visible fragments really were the ones that indicate a positive clone. For the further cloning, the plasmids were isolated and digested directly.</p> | <p>Some samples of the new colony PCR showed bands slightly higher than 750 bp, which would resemble the size of the expected fragment of ca 800 bp, but since the controls with the nose plasmids containing the Ag sensitive promoter (all confirmed positive by control PCR on isolated plasmids) were not all positive, it was uncertain, that the visible fragments really were the ones that indicate a positive clone. For the further cloning, the plasmids were isolated and digested directly.</p> | ||
</div> | </div> | ||
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</fieldset> | </fieldset> | ||
<fieldset class="exp23"> | <fieldset class="exp23"> | ||
- | <legend><a name="exp23.48">23.48 Purification of | + | <legend><a name="exp23.48">23.48 Purification of digested plasmid and fragments</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: In order to improve the ligation results pure DNA samples are needed</p> | <p>Aim: In order to improve the ligation results pure DNA samples are needed</p> | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>All | + | <p>All digested samples have been purified via gel extraction, resulting in the following concentration:</p> |
<ul class="list"> | <ul class="list"> | ||
<li>pSB1C3= 19 ng/µl</li> | <li>pSB1C3= 19 ng/µl</li> | ||
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<legend><a name="exp13.115">13.115 Ligation of piGEM008/009 with constitutive promoter</a></legend> | <legend><a name="exp13.115">13.115 Ligation of piGEM008/009 with constitutive promoter</a></legend> | ||
<div class="aim"> | <div class="aim"> | ||
- | <p>Aim: ligate the | + | <p>Aim: ligate the digested vector with the constitutive promoter</p> |
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
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</table> | </table> | ||
<br /> | <br /> | ||
- | <p>Gel analysis of the | + | <p>Gel analysis of the digested plasmids reveals that all clones are negative, new ligation will be performed with a higher amount of insert.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The plasmids were isolated from the inoculated cultures (13.115; 2 clones piGEM002 + const, 6 clones piGEM007 + const, 5 clones piGEM008 + const and 4 clones piGEM009 + const) and | + | <p>The plasmids were isolated from the inoculated cultures (13.115; 2 clones piGEM002 + const, 6 clones piGEM007 + const, 5 clones piGEM008 + const and 4 clones piGEM009 + const) and digested with the enzymes <i>Nco</i>I-HF and <i>Eco</i>RI-HF. Negative plasmids should yield fragments in size of 476 and 6190 (for piGEM002) or 6235 bp (for piGEM007/008/009). The positive plasmids should result in fragments in the size of 711 and 6190/6235 bp.</p> |
<br /> | <br /> | ||
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
Line 4,487: | Line 4,487: | ||
<p>The ligation was performed at 16°C for 3 hours. | <p>The ligation was performed at 16°C for 3 hours. | ||
<br /> | <br /> | ||
- | <p>After the ligation the reaction was inactivated by incubating the reaction mix at | + | <p>After the ligation the reaction was inactivated by incubating the reaction mix at 85°C for 10 minutes. Afterwards chemically competent <i>E. coli</i> XL-1-blue were transformed with the whole reaction mix. Standard <i>E. coli</i> transformation protocol was used. |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
Line 4,501: | Line 4,501: | ||
<legend><a name="exp13.118">13.118 control restriction of piGEM009 with the constitutive promoter</a></legend> | <legend><a name="exp13.118">13.118 control restriction of piGEM009 with the constitutive promoter</a></legend> | ||
<div class="aim"> | <div class="aim"> | ||
- | <p>Aim: check the plasmid for the | + | <p>Aim: check the plasmid for the correct insertion of the promoter</p> |
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The plasmids were isolated from the cultures (13.117) and | + | <p>The plasmids were isolated from the cultures (13.117) and digested again with the enzymes <i>Nco</i>I and <i>Eco</i>RI. The expected fragments were the same like in 13.117.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> |