Difference between revisions of "Team:Pasteur Paris/Microbiology week2"
| (7 intermediate revisions by 2 users not shown) | |||
| Line 48: | Line 48: | ||
padding-top:1%; | padding-top:1%; | ||
margin-left:10%; | margin-left:10%; | ||
| + | } | ||
| + | |||
| + | h6 { | ||
| + | display : block; | ||
| + | font-family: 'Oswald', Arial, sans-serif; | ||
| + | line-height : 1.5; | ||
} | } | ||
| Line 162: | Line 168: | ||
position: relative; | position: relative; | ||
text-shadow: 0 1px 0 #000; | text-shadow: 0 1px 0 #000; | ||
| + | text-align : center; | ||
} | } | ||
| Line 174: | Line 181: | ||
top: 25%; | top: 25%; | ||
width: 100%; | width: 100%; | ||
| + | text-align : center; | ||
} | } | ||
| Line 229: | Line 237: | ||
<p><h3><B> June 14, 2016:</B></h3></p> | <p><h3><B> June 14, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp4"><h4> 8. Make a culture of DH5α pET43.1 to monitor the growth curve </h4></a></br> | + | <a href="#exp4"><h4> 8. Make a culture of DH5α pET43.1(a+) to monitor the growth curve </h4></a></br> |
</p> | </p> | ||
| Line 257: | Line 265: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U>Aim:</U> To check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.</br> Seed pET43.1 DH5α in LB + carbenicillin media to make stab culture. Indeed, the validity of transformants needs to be verified by checking if an insert is present in the transformed plasmid. Secondly, verified plasmid-DH5&alpha combinations need to be stored beyond the lifetime of plate colonies. </br></br> | + | <h6><U>Aim:</U></h6> To check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.</br> |
| − | + | Seed pET43.1 DH5α in LB + carbenicillin media to make stab culture. Indeed, the validity of transformants needs to be verified by checking if an insert is present in the transformed plasmid. Secondly, verified plasmid-DH5&alpha combinations need to be stored beyond the lifetime of plate colonies. </br></br><br /> | |
| − | <U>What we did in the lab:</U></br>Counting of colonies on petri dish: overnight grown plates at 37°C, supplemented with carbenicillin 50 µg/ml (LB + CB50) or with chloramphenicol 34 µg/ml (LB + CM34) were counted for the presence of individual distinct colonies. </br></br>LB + CB50</br></br> | + | <h6><U>What we did in the lab:</U></h6></br>Counting of colonies on petri dish: overnight grown plates at 37°C, supplemented with carbenicillin 50 µg/ml (LB + CB50) or with chloramphenicol 34 µg/ml (LB + CM34) were counted for the presence of individual distinct colonies. </br></br>LB + CB50</br></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 3 :</U> LB + CM34</p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 273: | Line 282: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+) DH5α</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+) DH5α</p></strong></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
| − | <td>23</td> | + | <td align = "center"; valign = "center">23</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>pET43.1a(+) DH5α</br> (different batch)</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>pET43.1a(+) DH5α</br> (different batch)</p></strong></td> |
| − | <td>Uncountable (Overgrown)</td> | + | <td align = "center"; valign = "center">Uncountable (Overgrown)</td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
| + | <td align = "center"; valign = "center">23</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+) DH5α</br> (different batch)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+) DH5α</br> (different batch)</p></strong></td> |
| − | <td>Uncountable </br>(Overgrown)</td> | + | <td align = "center"; valign = "center">Uncountable </br>(Overgrown)</td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
| + | <td align = "center"; valign = "center">23</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | </br> | + | </br><br /> |
| − | < | + | |
| + | |||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 4</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 304: | Line 317: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pSB1C3 DH5α</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pSB1C3 DH5α</p></strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>78</td> | + | <td align = "center"; valign = "center">78</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>pSB1C3 DH5α</br> (different batch)</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>pSB1C3 DH5α</br> (different batch)</p></strong></td> |
| − | <td>38 </td> | + | <td align = "center"; valign = "center">38 </td> |
| − | <td> Uncountable (Overgrown) </td> | + | <td align = "center"; valign = "center"> Uncountable (Overgrown) </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table></br> |
| − | </br> | + | </br><br /><br /> |
| − | < | + | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 329: | Line 341: | ||
<a href="# exp2" class="closemsg"></a> | <a href="# exp2" class="closemsg"></a> | ||
<figcaption><p> | <figcaption><p> | ||
| − | <U>What we did in the lab:</U></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a></br></br><br /> |
| − | <U>Materials:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| + | <h6><U>Materials:</U></h6> | ||
• 1X TAE buffer </br> | • 1X TAE buffer </br> | ||
| − | • Agarose <br> | + | • Agarose </br> |
| − | • Microwave oven | + | • Microwave oven </br> |
• Distilled water</br> | • Distilled water</br> | ||
• Ethidium Bromide</br> | • Ethidium Bromide</br> | ||
• Gel caster, and power supply</br> | • Gel caster, and power supply</br> | ||
| − | • Precision balance </br> | + | • Precision balance </br></br> |
| − | + | <h6><U>Method:</U></h6> | |
| − | </br> | + | 1. Buffer solution: we have 1.0X stock TAE (Tris Acetate EDTA) solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer. </br> |
| − | <U>Method:</U></ | + | |
| − | 1. Buffer solution: we have 1.0X stock TAE (Tris Acetate EDTA) solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer </br> | + | |
2. Agarose gel: We prepared 0.7% w/v agarose in 1X TAE and allow it to set.</br></br> | 2. Agarose gel: We prepared 0.7% w/v agarose in 1X TAE and allow it to set.</br></br> | ||
| Line 347: | Line 358: | ||
<I>Samples:</I></br> | <I>Samples:</I></br> | ||
• pSB1C3 plasmid (obtained with Midiprep on June 8, 2016)</br> | • pSB1C3 plasmid (obtained with Midiprep on June 8, 2016)</br> | ||
| − | • pSB1C3 plasmid (digested by SpeI/XbaI)<br> | + | • pSB1C3 plasmid (digested by SpeI/XbaI)<br /> |
• pET43.1a plasmid (obtained with Midiprep on June 8, 2016)< /br> | • pET43.1a plasmid (obtained with Midiprep on June 8, 2016)< /br> | ||
• pET43.1a plasmid digested by BamH I/Hind III</br> | • pET43.1a plasmid digested by BamH I/Hind III</br> | ||
| Line 360: | Line 371: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 5</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 376: | Line 388: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Sample </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Sample </p></strong></td> |
| − | <td>Molecular </br> weight</td> | + | <td align = "center"; valign = "center">Molecular </br> weight</td> |
| − | <td>pET43.1a(+)</br> uncut</td> | + | <td align = "center"; valign = "center">pET43.1a(+)</br> uncut</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pET43.1a(+)</br> B-H</td> | + | <td align = "center"; valign = "center">pET43.1a(+)</br> B-H</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pBS1C3</br> S-X</td> | + | <td align = "center"; valign = "center">pBS1C3</br> S-X</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pBS1C3</br> uncut</td> | + | <td align = "center"; valign = "center">pBS1C3</br> uncut</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> </strong></td> | + | <td align = "center"; valign = "center"><strong> </strong></td> |
| − | <td>DNA (µl)</td> | + | <td align = "center"; valign = "center">DNA (µl)</td> |
| − | <td> 5</td> | + | <td align = "center"; valign = "center"> 5</td> |
| − | <td> 2</td> | + | <td align = "center"; valign = "center"> 2</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 2</td> | + | <td align = "center"; valign = "center"> 2</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 4</td> | + | <td align = "center"; valign = "center"> 4</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 4</td> | + | <td align = "center"; valign = "center"> 4</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> </strong></td> | + | <td align = "center"; valign = "center"><strong> </strong></td> |
| − | <td> | + | <td align = "center"; valign = "center">H<sub>2</sub>0</td> |
| − | <td> 0</td> | + | <td align = "center"; valign = "center"> 0</td> |
| − | <td> 8</td> | + | <td align = "center"; valign = "center"> 8</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>8</td> | + | <td align = "center"; valign = "center">8</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>6</td> | + | <td align = "center"; valign = "center">6</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>6</td> | + | <td align = "center"; valign = "center">6</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> </strong></td> | + | <td align = "center"; valign = "center"><strong> </strong></td> |
| − | <td>6X</br>Loading</br>buffer</td> | + | <td align = "center"; valign = "center">6X</br>Loading</br>buffer</td> |
| − | <td> 0</td> | + | <td align = "center"; valign = "center"> 0</td> |
| − | <td> 2</td> | + | <td align = "center"; valign = "center"> 2</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table></br> |
| − | </br> | + | </br></br> |
| − | + | <h6><U>Results</U></h6></br> | |
| − | <U>Results</U></br> | + | The digestion has worked since the plasmids digested have moved faster. </br></br> |
| − | The digestion has worked since the plasmids digested have moved faster. </br></br>NB: the gel suffered from overheating. </br> | + |   NB: the gel suffered from overheating. </br> |
| − | </br> | + | </br></br></br> |
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 442: | Line 453: | ||
<a href="# exp3" class="closemsg"></a> | <a href="# exp3" class="closemsg"></a> | ||
<figcaption><p> | <figcaption><p> | ||
| − | <U>Aim:</U> Repeat Midiprep for pET43.1a(+). </br>Since we need a lot of pET43.1a(+) we have to repeat the Midiprep.</br></br> | + | <h6><U>Aim:</U></h6> Repeat Midiprep for pET43.1a(+). </br>Since we need a lot of pET43.1a(+) we have to repeat the Midiprep.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
• Falcon of 50 ml</br> | • Falcon of 50 ml</br> | ||
• Petri dish with DH5α transformed with pET43.1a(+)<br> | • Petri dish with DH5α transformed with pET43.1a(+)<br> | ||
• LB (Luria Broth) medium autoclaved (5 ml) </br> | • LB (Luria Broth) medium autoclaved (5 ml) </br> | ||
• Shaking incubator (INFORS HT)</br> | • Shaking incubator (INFORS HT)</br> | ||
| − | • Antibiotic: carbenicillin 100 mg/ml<br> | + | • Antibiotic: carbenicillin 100 mg/ml<br /> |
• Pipetman P10 and 5 ml graduated pipet</br> | • Pipetman P10 and 5 ml graduated pipet</br> | ||
• Bunsen burner </br> | • Bunsen burner </br> | ||
| − | • Cones for P10<br> | + | • Cones for P10<br /> |
• Propipet</br> | • Propipet</br> | ||
• Rack for 50 ml tubes </br></br> | • Rack for 50 ml tubes </br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. Pick one colony and put it into 5 ml of LB medium + 2.5 µl of carbenicillin stock, to obtain 50 µg/µl. | 1. Pick one colony and put it into 5 ml of LB medium + 2.5 µl of carbenicillin stock, to obtain 50 µg/µl. | ||
</br> | </br> | ||
2. Grow overnight at 37°C in a shaking incubator at 150 rpm.</br> | 2. Grow overnight at 37°C in a shaking incubator at 150 rpm.</br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| Line 478: | Line 487: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Use the preculture of June 13, 2016 to start the new culture.</br></br> | + | <h6><U> Aim:</U></h6> Use the preculture of June 13, 2016 to start the new culture.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
• Falcon of 50 ml</br> | • Falcon of 50 ml</br> | ||
| − | • LB autoclaved 500 ml<br> | + | • LB autoclaved 500 ml<br /> |
• 3 precultures performed on June 13, 2016 (redundant cultures in case colonies don’t grow)</br> | • 3 precultures performed on June 13, 2016 (redundant cultures in case colonies don’t grow)</br> | ||
• carbenicillin 100 mg/ml</br> | • carbenicillin 100 mg/ml</br> | ||
| − | • swing bucket centrifuge (JOUAN GR4i)</br> | + | • swing bucket centrifuge (JOUAN GR4i)</br></br> |
| − | + | <h6><U>Method:</U></h6> | |
| − | </br> | + | 1. Get back preculture performed on the June 13, 2016, put it in the swing bucket centrifuge during 7 minutes (3500 RPM) and remove the supernatant. </br> |
| − | <U>Method:</U></ | + | 2. Add 5ml of fresh LB, resuspend the pellet and centrifuge again at 3500 rpm during 7 minutes. </br> |
| − | 1. Get back preculture performed on the June 13, 2016, put it in the swing bucket centrifuge during 7 | + | |
| − | 2. Add 5ml of fresh LB, resuspend the pellet and centrifuge again at 3500 | + | |
3. Repeat one more time step 2.</br> | 3. Repeat one more time step 2.</br> | ||
| − | 4. Resuspend the pellet into 5 | + | 4. Resuspend the pellet into 5 ml of fresh LB+ 2.5 µl of carbenicillin 100 mg/ml.</br> |
| − | 5. Grow in the shaking incubator (T= 37 °C / 130 | + | 5. Grow in the shaking incubator (T= 37 °C / 130 rpm) overnight.</br> |
</br></br> | </br></br> | ||
We overshot the 0.7 OD, set point therefore we continued overnight. </br> | We overshot the 0.7 OD, set point therefore we continued overnight. </br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 512: | Line 520: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> To make a stab culture we need to capture the bacteria growing at the exponential phase </br> In order to do that we need to take optical density at 600<sub>nm</sub> (OD600<sub>nm</sub>).</br></br> | + | <h6><U> Aim:</U></h6> To make a stab culture we need to capture the bacteria growing at the exponential phase. </br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | In order to do that we need to take optical density at 600<sub>nm</sub> (OD600<sub>nm</sub>).</br></br> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/b/b1/Stab_culture_Pasteur_Paris_2016.pdf">link</a></br></br><br /> |
| − | <U>Materials:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| + | <h6><U>Materials:</U></h6> | ||
• Falcon of 50 ml </br> | • Falcon of 50 ml </br> | ||
• LB autoclaved 500 ml<br> | • LB autoclaved 500 ml<br> | ||
| Line 521: | Line 530: | ||
&bull Precultures performed on the June 14, 2016</br> | &bull Precultures performed on the June 14, 2016</br> | ||
• Antibiotic: carbenicillin 100 mg/ml</br> | • Antibiotic: carbenicillin 100 mg/ml</br> | ||
| − | • Microbiology equipment</br> | + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>)</br></br> |
| − | + | <h6><U>Method:</U></h6> Before starting the culture we need to have molten LB-agar so we need to place the solid LB agar on a hot plate with stirring. </br> | |
| − | </br> | + | 1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at OD600<sub>nm</sub> every hour for the first three hours, then every 20 minutes onwards. We recorded OD600<sub>nm</sub> for this specific experiment after 3 hours.</br></br> |
| − | <U>Method:</U></ | + | |
| − | 1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at OD600<sub>nm</sub> every hour for the first three hours, then every 20 | + | |
| − | </br> | + | |
| − | + | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 6</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 540: | Line 546: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>12h37 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>12h37 </p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>0.013</td> | + | <td align = "center"; valign = "center">0.013</td> |
| − | <td>0.015</td> | + | <td align = "center"; valign = "center">0.015</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>13h07</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>13h07</p></strong></td> |
| − | <td>0.023</td> | + | <td align = "center"; valign = "center">0.023</td> |
| − | <td> 0.027</td> | + | <td align = "center"; valign = "center"> 0.027</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>13h37</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>13h37</p></strong></td> |
| − | <td>0.063</td> | + | <td align = "center"; valign = "center">0.063</td> |
| − | <td> 0.059 </td> | + | <td align = "center"; valign = "center"> 0.059 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>14h07</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>14h07</p></strong></td> |
| − | <td>0.110 </td> | + | <td align = "center"; valign = "center">0.110 </td> |
| − | <td> 0.105</td> | + | <td align = "center"; valign = "center"> 0.105</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>14h27</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>14h27</p></strong></td> |
| − | <td>0.172 </td> | + | <td align = "center"; valign = "center">0.172 </td> |
| − | <td> 0.170</td> | + | <td align = "center"; valign = "center"> 0.170</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>14h47</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>14h47</p></strong></td> |
| − | <td>0.265 </td> | + | <td align = "center"; valign = "center">0.265 </td> |
| − | <td> 0.256</td> | + | <td align = "center"; valign = "center"> 0.256</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>15h07</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>15h07</p></strong></td> |
| − | <td>0.384 </td> | + | <td align = "center"; valign = "center">0.384 </td> |
| − | <td> 0.377 </td> | + | <td align = "center"; valign = "center"> 0.377 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>15h27</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>15h27</p></strong></td> |
| − | <td>0.491 </td> | + | <td align = "center"; valign = "center">0.491 </td> |
| − | <td> 0.482</td> | + | <td align = "center"; valign = "center"> 0.482</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>15h47</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>15h47</p></strong></td> |
| − | <td>0.567 </td> | + | <td align = "center"; valign = "center">0.567 </td> |
| − | <td> 0.557</td> | + | <td align = "center"; valign = "center"> 0.557</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>16h06</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>16h06</p></strong></td> |
| − | <td>0.697</td> | + | <td align = "center"; valign = "center">0.697</td> |
| − | <td> 0.608</td> | + | <td align = "center"; valign = "center"> 0.608</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>16h29</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>16h29</p></strong></td> |
| − | <td>0.816</td> | + | <td align = "center"; valign = "center">0.816</td> |
| − | <td> 0.755</td> | + | <td align = "center"; valign = "center"> 0.755</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>17h03</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>17h03</p></strong></td> |
| − | <td>0.954 </td> | + | <td align = "center"; valign = "center">0.954 </td> |
| − | <td> 0.909 </td> | + | <td align = "center"; valign = "center"> 0.909 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>17h20</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>17h20</p></strong></td> |
| − | <td>0.954</td> | + | <td align = "center"; valign = "center">0.954</td> |
| − | <td> 0.909</td> | + | <td align = "center"; valign = "center"> 0.909</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>17h40</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>17h40</p></strong></td> |
| − | <td>1.061</td> | + | <td align = "center"; valign = "center">1.061</td> |
| − | <td> 1.029</td> | + | <td align = "center"; valign = "center"> 1.029</td> |
</tr | </tr | ||
<tr> | <tr> | ||
| − | <td><strong> <p>18h00</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>18h00</p></strong></td> |
| − | <td>1.092</td> | + | <td align = "center"; valign = "center">1.092</td> |
| − | <td> 1.082</td> | + | <td align = "center"; valign = "center"> 1.082</td> |
</tr | </tr | ||
<tr> | <tr> | ||
| − | <td><strong> <p>18h00</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>18h00</p></strong></td> |
| − | <td>1.122</td> | + | <td align = "center"; valign = "center">1.122</td> |
| − | <td> 1.132</td> | + | <td align = "center"; valign = "center"> 1.132</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | </br> | + | </br><br /><br /> |
| − | < | + | |
| − | < | + | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
| Line 636: | Line 638: | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
| Line 643: | Line 646: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | < | + | |
| − | <U> What we did on the lab:</U></br> | + | <h6><U> What we did on the lab:</U></h6></br> |
| − | <U> Method:</U></ | + | <h6><U> Method:</U></h6> |
1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 µl of carbenicillin 50 mg/ml added, swirl to mix.</br> | 1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 µl of carbenicillin 50 mg/ml added, swirl to mix.</br> | ||
2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total). </br> | 2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total). </br> | ||
3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times.</br> | 3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times.</br> | ||
4. Grow overnight at 37 °C and next day place it at 4°C.</br> | 4. Grow overnight at 37 °C and next day place it at 4°C.</br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
| − | |||
</div> | </div> | ||
| Line 663: | Line 666: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> We received our gene block synthesized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer. </br></br> | + | <h6><U> Aim:</U></h6> We received our gene block synthesized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer. </br></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> Add TE (Tris 10 mM pH 8.0 EDTA 1 mM) buffer in each tubes and refer to the next table for volumes. |
</br></br> | </br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 7</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 679: | Line 683: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A1 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A1 </p></strong></td> |
| − | <td>500</td> | + | <td align = "center"; valign = "center">500</td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">50</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A2 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A2 </p></strong></td> |
| − | <td>500</td> | + | <td align = "center"; valign = "center">500</td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">50</td> |
| + | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C1 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C1 </p></strong></td> |
| − | <td>1000</td> | + | <td align = "center"; valign = "center">1000</td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td>100</td> | + | <td align = "center"; valign = "center">100</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C2 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C2 </p></strong></td> |
| − | <td>1000</td> | + | <td align = "center"; valign = "center">1000</td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td>100</td> | + | <td align = "center"; valign = "center">100</td> |
</tr> | </tr> | ||
| − | <td><strong><p>D1 </p></strong></td> | + | <tr> |
| − | <td>500</td> | + | <td align = "center"; valign = "center"><strong><p>D1 </p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">500</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">10</td> |
| + | <td align = "center"; valign = "center">50</td> | ||
</tr> | </tr> | ||
| − | <td><strong><p>D2 </p></strong></td> | + | <tr> |
| − | <td>500</td> | + | <td align = "center"; valign = "center"><strong><p>D2 </p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">500</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">10</td> |
| + | <td align = "center"; valign = "center">50</td> | ||
</tr> | </tr> | ||
| − | <td><strong><p>E1 </p></strong></td> | + | <tr> |
| − | <td>500</td> | + | <td align = "center"; valign = "center"><strong><p>E1 </p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">500</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">10</td> |
| + | <td align = "center"; valign = "center">50</td> | ||
</tr> | </tr> | ||
| − | <td><strong><p>E2 </p></strong></td> | + | <tr> |
| − | <td>500</td> | + | <td align = "center"; valign = "center"><strong><p>E2 </p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">500</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">10</td> |
| + | <td align = "center"; valign = "center">50</td> | ||
</tr> | </tr> | ||
| − | <td><strong><p>B2 </p></strong></td> | + | <tr> |
| − | <td>1000</td> | + | <td align = "center"; valign = "center"><strong><p>B2 </p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">1000</td> |
| − | <td>100</td> | + | <td align = "center"; valign = "center">10</td> |
| + | <td align = "center"; valign = "center">100</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table></br> |
| − | </br> | + | </br><br /><br /> |
| − | < | + | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 737: | Line 746: | ||
| − | |||
| − | |||
<div class="lightbox" id="exp8"> | <div class="lightbox" id="exp8"> | ||
| Line 745: | Line 752: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> We want to have only the DNA backbone of pET43.1a(+) and pSB1C3, not the inserts that lie between the restriction sites. Spe I/ Xba I or BamH I/Hind III respectively, so we have to extract the plasmid backbone on out of an agarose gel. Furthermore, to clean the pET43.1a(+) and pSB1C3 backbones we use the gel extraction kit of QIAGEN. Afterwards we ligated our plasmid with the inserts. In this experiment we use inserts C1 and C2. | + | <h6><U> Aim:</U></h6> We want to have only the DNA backbone of pET43.1a(+) and pSB1C3, not the inserts that lie between the restriction sites. Spe I/ Xba I or BamH I/Hind III respectively, so we have to extract the plasmid backbone on out of an agarose gel. Furthermore, to clean the pET43.1a(+) and pSB1C3 backbones we use the gel extraction kit of QIAGEN. Afterwards we ligated our plasmid with the inserts. In this experiment we use inserts C1 and C2. |
</br></br> | </br></br> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4e/T--Pasteur_Paris--Gel_extraction_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Material:</U></ | + | <h6><U>Material:</U></h6> |
| − | • | + | • NEB extraction kit. Monarch DNA Gel Extraction Kit (/#T1020G). </br></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. Excise the DNA fragment from the agarose gel and transfer to 1 ml Eppendorf tube. </br> | 1. Excise the DNA fragment from the agarose gel and transfer to 1 ml Eppendorf tube. </br> | ||
| Line 759: | Line 766: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 8</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 771: | Line 779: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+)</p></strong></td> |
| − | <td>m1</td> | + | <td align = "center"; valign = "center">m1</td> |
| − | <td>0.9</td> | + | <td align = "center"; valign = "center">0.9</td> |
| − | <td>1.3</td> | + | <td align = "center"; valign = "center">1.3</td> |
| − | <td>329.6</td> | + | <td align = "center"; valign = "center">329.6</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+)</p></strong></td> |
| − | <td>m2</td> | + | <td align = "center"; valign = "center">m2</td> |
| − | <td>0.9</td> | + | <td align = "center"; valign = "center">0.9</td> |
| − | <td>1.3</td> | + | <td align = "center"; valign = "center">1.3</td> |
| − | <td>265.3</td> | + | <td align = "center"; valign = "center">265.3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pSB1C3 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pSB1C3 </p></strong></td> |
| − | <td>m1</td> | + | <td align = "center"; valign = "center">m1</td> |
| − | <td>0.9</td> | + | <td align = "center"; valign = "center">0.9</td> |
| − | <td>1.2</td> | + | <td align = "center"; valign = "center">1.2</td> |
| − | <td>251.1</td> | + | <td align = "center"; valign = "center">251.1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> pSB1C3 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> pSB1C3 </p></strong></td> |
| − | <td>m2</td> | + | <td align = "center"; valign = "center">m2</td> |
| − | <td>1.0</td> | + | <td align = "center"; valign = "center">1.0</td> |
| − | <td>1.2</td> | + | <td align = "center"; valign = "center">1.2</td> |
| − | <td>185.6</td> | + | <td align = "center"; valign = "center">185.6</td> |
| + | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | </br></br> | |
| − | + | ||
3. Incubate the samples between 37-55°C, vortexing periodically until the gel slice is completely dissolved (/ ~ 10 min). </br> | 3. Incubate the samples between 37-55°C, vortexing periodically until the gel slice is completely dissolved (/ ~ 10 min). </br> | ||
| − | 4. Load sample onto the column. Close the cap, spin for 1 | + | 4. Load sample onto the column. Close the cap, spin for 1 minute then discard flow through.</br> |
5. Re insert column into collection tube. Add 200 µµl DNA wash buffer and spin for one minute. Discard flow-through.</br> | 5. Re insert column into collection tube. Add 200 µµl DNA wash buffer and spin for one minute. Discard flow-through.</br> | ||
6. Repeat step 5.</br> | 6. Repeat step 5.</br> | ||
7. Transfer column to a clean 1.5 ml microfuge tube.</br> | 7. Transfer column to a clean 1.5 ml microfuge tube.</br> | ||
| − | 8. Add 6 µl of DNA Elution buffer to the center of the matric. Wait for 1 | + | 8. Add 6 µl of DNA Elution buffer to the center of the matric. Wait for 1 minute and spin for 1 minute to elute DNA.</br> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| Line 826: | Line 831: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> After digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2. </br></br> | + | <h6><U> Aim:</U></h6> After digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2. </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U>< | + | <h6><U>What we did in the lab:</U><h6></br> |
| − | + | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | 1. Add all reagents in 1 ml Eppendorf. | + | 1. Add all reagents in 1 ml Eppendorf. </br> |
| − | 2. Digest during 2 h at 37 °C. | + | 2. Digest during 2 h at 37 °C. </br> |
| − | 3. For the reagent volumes, refer to the Table 9. Total volume = 50 µl. | + | 3. For the reagent volumes, refer to the Table 9. Total volume = 50 µl.</br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 9</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| − | |||
<th></th> | <th></th> | ||
<th>C1</th> | <th>C1</th> | ||
| Line 849: | Line 854: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></td> |
| − | <td>20</td> | + | <td align = "center"; valign = "center">20</td> |
| − | <td>20</td> | + | <td align = "center"; valign = "center">20</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>BamH I (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>BamH I (µl)</p></strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td | + | <td align = "center"; valign = "center">1</td> |
| − | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Hind III (µl)</ </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Hind III (µl)</ </p></strong></td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
| − | <td>2</td | + | <td align = "center"; valign = "center">2</td |
</tr> | </tr> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> H<sub>2</sub>0 (µl)</ </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> H<sub>2</sub>0 (µl)</ </p></strong></td> |
| − | <td>22</td> | + | <td align = "center"; valign = "center">22</td> |
| − | <td>22</td | + | <td align = "center"; valign = "center">22</td> |
| − | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> CutSmart(µl)</ </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> CutSmart(µl)</ </p></strong></td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | </br> | + | </br></br> |
| − | < | + | <br /><br /><br /> |
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| − | + | ||
| + | |||
<div class="lightbox" id="exp10"> | <div class="lightbox" id="exp10"> | ||
| Line 895: | Line 898: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | < | + | <h6><U> Aim:</U></h6> Dephosphorylate our inserts</br></br> |
| − | + | ||
| − | + | ||
| + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/c/c0/T--Pasteur_Paris--dephosphorylation_protocol.pdf">link</a></br></br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| − | + | ||
| + | |||
| + | |||
<div class="lightbox" id="exp11"> | <div class="lightbox" id="exp11"> | ||
<figure> | <figure> | ||
| Line 909: | Line 914: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U | + | <h6><U> Aim:</U></h6> Resuspend C1, C2 and all inserts synthesized by iDT. <br /><br /> |
| − | + | ||
| − | + | ||
| + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a></br></br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| − | + | ||
</body> | </body> | ||
| − | <html> | + | </html> |
Latest revision as of 21:35, 19 October 2016
