Difference between revisions of "Team:Pasteur Paris/Microbiology week4"
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> We want to extract the DNA we tried to transform on June 22, 2016.<br> | + | <h6><U> Aim:</U></h6> We want to extract the DNA we tried to transform on June 22, 2016.<br> |
The aim is to obtain the construct of C1 in pET43.1 for expression in <i>E. coli</i>. This to serve both the verification of the presence of the pET43.1-C1 plasmid in the cells but also to make a stock of it for future cloning and expression experiments. | The aim is to obtain the construct of C1 in pET43.1 for expression in <i>E. coli</i>. This to serve both the verification of the presence of the pET43.1-C1 plasmid in the cells but also to make a stock of it for future cloning and expression experiments. | ||
</br></br> | </br></br> | ||
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a></br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a></br></br> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Assay the DNA to check if the Midiprep was efficient, and to have the concentration of pET43.1-C1 for transformations and sequencing.</br> </br> | + | <p> |
| − | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | + | <h6><U> Aim:</U></h6> Assay the DNA to check if the Midiprep was efficient, and to have the concentration of pET43.1-C1 for transformations and sequencing.</br> </br> |
| − | <U>What we did in the lab:</U></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>Material:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | •pET43.1-C1 obtained by the midiprep kit | + | <h6><U>Material:</U></h6> |
| − | •plastic uvette (Eppendorf) | + | •pET43.1-C1 obtained by the midiprep kit</br> |
| − | •spectrophotometer | + | •plastic uvette (Eppendorf)</br> |
| − | •TE (Tris 10 mM pH 8.0, EDTA 1 mM) buffer | + | •spectrophotometer </br> |
| + | •TE (Tris 10 mM pH 8.0, EDTA 1 mM) buffer</br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | 1. Use a 1/50 dilution of pET43.1-C1 plasmid in TE, volume =500 µl</br> | + | 1. Use a 1/50 dilution of pET43.1-C1 plasmid in TE, volume =500 µl.</br> |
| − | 2. We use the spectrophotometer ultrospec 3100 pro- Amersham Bioscience and we do the blank with TE</br> | + | 2. We use the spectrophotometer ultrospec 3100 pro- Amersham Bioscience and we do the blank with TE.</br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 18 :</U> absorbance of DNA measured at 260<sub>nm</sub>, and using ds-DNA (double stranded DNA) absorbance of 50 µg/ml for an optical density of 1 (A260<sub>nm</sub>=1)</p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 315: | Line 323: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p><span>A 260</span> nm</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p><span>A 260</span> nm</p></strong></td> |
| − | <td>0.065</td> | + | <td align = "center"; valign = "center">0.065</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p><span>A 280</span> nm </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p><span>A 280</span> nm </p></strong></td> |
| − | <td>0.57</td> | + | <td align = "center"; valign = "center">0.57</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p><span>A 260</span> nm/<strong><p>A<span>280 nm</span></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p><span>A 260</span> nm/<strong><p>A<span>280 nm</span></p></strong></td> |
| − | <td>1.14</td> | + | <td align = "center"; valign = "center">1.14</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinal </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinal </p></strong></td> |
| − | <td>1.14 ng/µl</td> | + | <td align = "center"; valign = "center">1.14 ng/µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /><br /> |
| − | + | </br></br></br> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
<div class="lightbox" id="exp3"> | <div class="lightbox" id="exp3"> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> : We need to verify that the pET43.1-C1 we isolated from transformation of the ligated products does indeed contain the C1 insert. In order to achieve that we will split the ligated vector into insert and the plasmid with restriction enzymes.</br> </br> | + | <h6><U> Aim:</U></h6> : We need to verify that the pET43.1-C1 we isolated from transformation of the ligated products does indeed contain the C1 insert. In order to achieve that we will split the ligated vector into insert and the plasmid with restriction enzymes.</br> </br> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a></br></br> | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Material:</U></ | + | <h6><U>Material:</U></h6> |
| − | •30 µl of pET43.C1 at 162 ng/µl</br> | + | •30 µl of pET43.C1 at 162 ng/µl</br><br /> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
n = C*V </br> | n = C*V </br> | ||
n = 162 * 30 = 4860 ng </br> | n = 162 * 30 = 4860 ng </br> | ||
We have 4860 ng and we want to digest 400 ng of DNA.</br> | We have 4860 ng and we want to digest 400 ng of DNA.</br> | ||
1. Add all reagent in a 1 ml Eppendorf</br> | 1. Add all reagent in a 1 ml Eppendorf</br> | ||
| − | 2. Allow the digestion to proceed during 1 h at | + | 2. Allow the digestion to proceed during 1 h at 37°C.</br> |
3. Incubate 5 min at 65°C to inactivate the enzymes.</br></br> | 3. Incubate 5 min at 65°C to inactivate the enzymes.</br></br> | ||
| − | <U> | + | <h6><U>Aim:</U></h6> We didn'tget any inserts, so we decided to repeat the preparation of pET43.1 and also the inserts C1 and C2.</br></br><br /> |
| − | <U> | + | <h6><U>What we did in the lab:</U></h6></br> |
| + | <h6><U>Materials:</U></h6> | ||
•pET43.1 at 86.7 ng/µl, </br> | •pET43.1 at 86.7 ng/µl, </br> | ||
•BamH I, Hind III, Cutsmart buffer, </br> | •BamH I, Hind III, Cutsmart buffer, </br> | ||
•all restriction reagents being from NEB (New England Biolabs).</br></br> | •all restriction reagents being from NEB (New England Biolabs).</br></br> | ||
| + | <h6><U>Method :</U></h6> | ||
| − | + | Prepare the mix composed of plasmid DNA, two enzymes, and buffer in an Eppendorf tube. </br><br /> | |
| − | Prepare the mix composed of plasmid DNA, two enzymes, and buffer in an Eppendorf tube. </br> | + | |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 19</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol DNA </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol DNA </p></strong></td> |
| − | <td>4.9 µl</td> | + | <td align = "center"; valign = "center">4.9 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol Hind III </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol Hind III </p></strong></td> |
| − | <td>1 µl</td> | + | <td align = "center"; valign = "center">1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol BamH I </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol BamH I </p></strong></td> |
| − | <td>0.5 µl</td> | + | <td align = "center"; valign = "center">0.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Vol H20 </p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Vol H20 </p></strong></td> |
| − | <td>11.6 µl</td> | + | <td align = "center"; valign = "center">11.6 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Vol Buffer (10X) Cutsmart</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Vol Buffer (10X) Cutsmart</p></strong></td> |
| − | <td>2 µl</td> | + | <td align = "center"; valign = "center">2 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Vol TOTAL</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Vol TOTAL</p></strong></td> |
| − | <td>20 µl</td> | + | <td align = "center"; valign = "center">20 µl</td> |
</tr> | </tr> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table></br> | + | </table></br></br> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
<div class="lightbox" id="exp4"> | <div class="lightbox" id="exp4"> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim :</U></h6> Digestion with new enzymes Xba I and Hind III of inserts (C1 and C2) and pET43.1a(+)</br> |
We have decided to switch from BamH I Hind III restriction digests to Xba I Hind III so that could remove the region containing a NusA fusion in the pET43.1a vector, as well as the following tags, and protease cleavage sites. This will make a cleaner construct for our protein.</br> </br> | We have decided to switch from BamH I Hind III restriction digests to Xba I Hind III so that could remove the region containing a NusA fusion in the pET43.1a vector, as well as the following tags, and protease cleavage sites. This will make a cleaner construct for our protein.</br> </br> | ||
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a></br></br> | + | <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> |
Prepare the digestion mix for the two inserts C1 and C2, as well as for the pET43.1 vector as in table 19 below.</br> | Prepare the digestion mix for the two inserts C1 and C2, as well as for the pET43.1 vector as in table 19 below.</br> | ||
Proceed with the digestion for 1h30 min.</br> | Proceed with the digestion for 1h30 min.</br> | ||
| Line 436: | Line 446: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 20</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 447: | Line 458: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol DNA </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol DNA </p></strong></td> |
| − | <td>23 µl</td> | + | <td align = "center"; valign = "center">23 µl</td> |
| − | <td>30 µl</td> | + | <td align = "center"; valign = "center">30 µl</td> |
| − | <td>40 µl</td> | + | <td align = "center"; valign = "center">40 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Xba I 20 000 U/ml (10 U) </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Xba I 20 000 U/ml (10 U) </p></strong></td> |
| − | <td>0.5 µl</td> | + | <td align = "center"; valign = "center">0.5 µl</td> |
| − | <td>0.5 µl</td> | + | <td align = "center"; valign = "center">0.5 µl</td> |
| − | <td>0.5 µl</td> | + | <td align = "center"; valign = "center">0.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Hind III 20 000 U/ml (20 U) </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Hind III 20 000 U/ml (20 U) </p></strong></td> |
| − | <td>1 µl</td> | + | <td align = "center"; valign = "center">1 µl</td> |
| − | <td>1 µl</td> | + | <td align = "center"; valign = "center">1 µl</td> |
| − | <td>1 µl</td> | + | <td align = "center"; valign = "center">1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Vol H<sub>2</sub>0 </p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Vol H<sub>2</sub>0 </p></strong></td> |
| − | <td>18.5 µl</td> | + | <td align = "center"; valign = "center">18.5 µl</td> |
| − | <td>13.5 µl</td> | + | <td align = "center"; valign = "center">13.5 µl</td> |
| − | <td> 3.5 µl</td> | + | <td align = "center"; valign = "center"> 3.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Vol Buffer (10X) Cutsmart</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Vol Buffer (10X) Cutsmart</p></strong></td> |
| − | <td>5 µl</td> | + | <td align = "center"; valign = "center">5 µl</td> |
| − | <td>5 µl</td> | + | <td align = "center"; valign = "center">5 µl</td> |
| − | <td>5 µl</td> | + | <td align = "center"; valign = "center">5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Vol TOTAL</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Vol TOTAL</p></strong></td> |
| − | <td>50 µl</td> | + | <td align = "center"; valign = "center">50 µl</td> |
| − | <td>50 µl</td> | + | <td align = "center"; valign = "center">50 µl</td> |
| − | <td>50 µl</td> | + | <td align = "center"; valign = "center">50 µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
1. Add all reagents in a 1 ml Eppendorf (1 for C1,1 for C2 and 1 for pET43.1)</br> | 1. Add all reagents in a 1 ml Eppendorf (1 for C1,1 for C2 and 1 for pET43.1)</br> | ||
2. Allow digestion to proceed for 1h30 at 37 °C then incubate 5 min at 65°C</br></br> | 2. Allow digestion to proceed for 1h30 at 37 °C then incubate 5 min at 65°C</br></br> | ||
| − | In Table | + | In Table 20: the inserts are those synthesized by iDT</br> |
Quantity of enzymes: </br> | Quantity of enzymes: </br> | ||
| Line 507: | Line 517: | ||
To save time, we prepared another 0.7% agarose gel that we stored at it 4°C with a wet towel containing TAE 1X (Tris-Acetate 10 mM, EDTA 1 mM).</br> | To save time, we prepared another 0.7% agarose gel that we stored at it 4°C with a wet towel containing TAE 1X (Tris-Acetate 10 mM, EDTA 1 mM).</br> | ||
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 519: | Line 529: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Determine the quantity of DNA we have after the gel extraction.</br> | + | <h6><U> Aim:</U></h6> Determine the quantity of DNA we have after the gel extraction.</br> |
| − | We will need to proceed with ligation of the inserts and vectors, and need to establish several ratios accurately.</br> </br> | + | We will need to proceed with ligation of the inserts and vectors, and need to establish several ratios accurately.</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> |
•a 50 µl capacity quartz cuvette</br> | •a 50 µl capacity quartz cuvette</br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
We use the spectrophotometer (Ultrospec 3100 pro).</br> | We use the spectrophotometer (Ultrospec 3100 pro).</br> | ||
| Line 540: | Line 550: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 21</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 552: | Line 563: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<sub>260 nm</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<sub>260 nm</sub></p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> A<sub>280 nm</sub> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> A<sub>280 nm</sub> </p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260 nm</span> /A<span>280 nm</span> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<span>260 nm</span> /A<span>280 nm</span> </p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> </td> | + | <td align = "center"; valign = "center"> </td> |
| − | <td>2.1</td> | + | <td align = "center"; valign = "center">2.1</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> |
| − | <td>2800 ng/µl </td> | + | <td align = "center"; valign = "center">2800 ng/µl </td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>2700 ng/µl </td> | + | <td align = "center"; valign = "center">2700 ng/µl </td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table></br></br> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 599: | Line 610: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Check if the digestion has been correctly done, if the two restriction site of the plasmid have been digested. We used the second gel we saved to re run the digestion products, as the measurements of the spectrophotometer were inconsistent with the amount of DNA we started with !</br></br> | + | <h6><U> Aim:</U></h6> Check if the digestion has been correctly done, if the two restriction site of the plasmid have been digested. We used the second gel we saved to re run the digestion products, as the measurements of the spectrophotometer were inconsistent with the amount of DNA we started with !</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a></br></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>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> |
•pET43.1a plasmid (obtained with Midiprep done 16/06/16)</br> | •pET43.1a plasmid (obtained with Midiprep done 16/06/16)</br> | ||
•pET43.1a plasmid cut by Hind III/Xba I</br> | •pET43.1a plasmid cut by Hind III/Xba I</br> | ||
| Line 612: | Line 623: | ||
•P10 pipet, P20 pipet, test tube 250 ml, electrophoresis BIORAD Mini-Sub Cell GT, 2 types of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge Eppendorf 5415D, </br></br> | •P10 pipet, P20 pipet, test tube 250 ml, electrophoresis BIORAD Mini-Sub Cell GT, 2 types of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge Eppendorf 5415D, </br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. fill the electrophoresis chamber with 0.5X TAE</br> | 1. fill the electrophoresis chamber with 0.5X TAE</br> | ||
2. add 10 µl of load buffer 6X in your samples to have 60 µl</br> | 2. add 10 µl of load buffer 6X in your samples to have 60 µl</br> | ||
3. Load samples in each lane with 20 µl of DNA</br></br> | 3. Load samples in each lane with 20 µl of DNA</br></br> | ||
| − | Following this deposit table:</br> | + | Following this deposit table:</br><b /> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 22</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 633: | Line 645: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p> Name</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> Name</p></strong></td> |
| − | <td> pET43.1a(+) uncut </td> | + | <td align = "center"; valign = "center"> pET43.1a(+) uncut </td> |
| − | <td> pET43.1a(+) X/H </td> | + | <td align = "center"; valign = "center"> pET43.1a(+) X/H </td> |
| − | <td> C1 X/B </td> | + | <td align = "center"; valign = "center"> C1 X/B </td> |
| − | <td> C2 X/B </td> | + | <td align = "center"; valign = "center"> C2 X/B </td> |
| − | <td> Marker weight </td> | + | <td align = "center"; valign = "center"> Marker weight </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> DNA (µl)</p></strong></td> |
| − | <td> 50 </td> | + | <td align = "center"; valign = "center"> 50 </td> |
| − | <td> 50 </td> | + | <td align = "center"; valign = "center"> 50 </td> |
| − | <td> 50 </td> | + | <td align = "center"; valign = "center"> 50 </td> |
| − | <td> 50 </td> | + | <td align = "center"; valign = "center"> 50 </td> |
| − | <td> 50 </td> | + | <td align = "center"; valign = "center"> 50 </td> |
</tr> | </tr> | ||
| − | <td><strong><p> | + | <td align = "center"; valign = "center"><strong><p> H<sub>20</sub> (µl)</p></strong></td> |
| − | <td> 0 </td> | + | <td align = "center"; valign = "center"> 0 </td> |
| − | <td> 0 </td> | + | <td align = "center"; valign = "center"> 0 </td> |
| − | <td> 0 </td> | + | <td align = "center"; valign = "center"> 0 </td> |
| − | <td> 0 </td> | + | <td align = "center"; valign = "center"> 0 </td> |
| − | <td> 0 </td> | + | <td align = "center"; valign = "center"> 0 </td> |
</tr> | </tr> | ||
| − | <td><strong><p> Buffer T6X (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> Buffer T6X (µl)</p></strong></td> |
| − | <td> 10 </td> | + | <td align = "center"; valign = "center"> 10 </td> |
| − | <td> 10 </td> | + | <td align = "center"; valign = "center"> 10 </td> |
| − | <td> 10 </td> | + | <td align = "center"; valign = "center"> 10 </td> |
| − | <td> 10 </td> | + | <td align = "center"; valign = "center"> 10 </td> |
| − | <td> 10 </td> | + | <td align = "center"; valign = "center"> 10 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
| − | <U>Results:</U></ | + | <h6><U>Results:</U></h6> |
Agarose gel (Refer to the Protocol)</br> | Agarose gel (Refer to the Protocol)</br> | ||
L1: pET43.1 uncut</br> | L1: pET43.1 uncut</br> | ||
| Line 678: | Line 689: | ||
| − | <center><img src=" https://static.igem.org/mediawiki/2016/d/d5/4.fig4_Pasteur.png" width="300px"; alt="">< | + | <center><img src=" https://static.igem.org/mediawiki/2016/d/d5/4.fig4_Pasteur.png" width="300px"; alt=""><i><p><U>Figure 4</U></p></i></center></br></br> |
| − | + | - We can see that for pET43.1 cut there are 2 bands (the first is pET43.1 digested partially, the second band is pET43.1 totally digested). So we get back bands of pET43.1 undigested, and we digested it again one more time.</br> | |
| − | - We can see that for pET43.1 cut there are 2 bands (the first is pET43.1 digested partially, the second band is pET43.1 totally digested). So we get back bands of pET43.1 undigested, and we digested it again one more time.</br></br></br> | + | </br></br><br /> |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 696: | Line 705: | ||
<p> | <p> | ||
| − | <U> Aim:</U> Get back our DNA digested and proceed with purification.</br> | + | <h6><U> Aim:</U></h6> Get back our DNA digested and proceed with purification.</br> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4e/T--Pasteur_Paris--Gel_extraction_protocol.pdf">link</a></br></br> | + | <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></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | <U>Method:</U></br> | + | <h6><U>Method:</U></h6></br> |
To extract DNA we use the Gel Extraction Kit from QIAGEN and we follow the different steps detailed in the kit.</br> | To extract DNA we use the Gel Extraction Kit from QIAGEN and we follow the different steps detailed in the kit.</br> | ||
| Line 706: | Line 715: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 23</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 718: | Line 728: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(pET43.1a(+) 1</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(pET43.1a(+) 1</p><strong></td> |
| − | <td>1.07 g </td> | + | <td align = "center"; valign = "center">1.07 g </td> |
| − | <td>1.52 g</td> | + | <td align = "center"; valign = "center">1.52 g</td> |
| − | <td>0.45 mg</td> | + | <td align = "center"; valign = "center">0.45 mg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(pET43.1a(+) 2</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(pET43.1a(+) 2</p></strong></td> |
| − | <td>1.07 g </td> | + | <td align = "center"; valign = "center">1.07 g </td> |
| − | <td>1.65 g</td> | + | <td align = "center"; valign = "center">1.65 g</td> |
| − | <td>0.58 mg</td> | + | <td align = "center"; valign = "center">0.58 mg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(C1)</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(C1)</p><strong></td> |
| − | <td>1.07 g </td> | + | <td align = "center"; valign = "center">1.07 g </td> |
| − | <td>1.66 g</td> | + | <td align = "center"; valign = "center">1.66 g</td> |
| − | <td>0.58 mg</td> | + | <td align = "center"; valign = "center">0.58 mg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(C2)</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(C2)</p><strong></td> |
| − | <td>1.07 g </td> | + | <td align = "center"; valign = "center">1.07 g </td> |
| − | <td>1.61 g</td> | + | <td align = "center"; valign = "center">1.61 g</td> |
| − | <td>0.54 mg</td> | + | <td align = "center"; valign = "center">0.54 mg</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
Two lanes were used for an extraction for the pET43.1 (pET43.1(1) and pET43.1(2)).</br> These volumes were too big to be contained in a 2 ml Eppendorf tube so we divided all in two. Finally, we had 8 tubes.</br></br> | Two lanes were used for an extraction for the pET43.1 (pET43.1(1) and pET43.1(2)).</br> These volumes were too big to be contained in a 2 ml Eppendorf tube so we divided all in two. Finally, we had 8 tubes.</br></br> | ||
| Line 763: | Line 772: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 24</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 774: | Line 784: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(pET43.1a(+) 1</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(pET43.1a(+) 1</p><strong></td> |
| − | <td>0.45 </td> | + | <td align = "center"; valign = "center">0.45 </td> |
| − | <td>1.3</td> | + | <td align = "center"; valign = "center">1.3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(pET43.1a(+) 2</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(pET43.1a(+) 2</p></strong></td> |
| − | <td>1.58 </td> | + | <td align = "center"; valign = "center">1.58 </td> |
| − | <td>1.8</td> | + | <td align = "center"; valign = "center">1.8</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(C1)</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(C1)</p><strong></td> |
| − | <td>0.59 </td> | + | <td align = "center"; valign = "center">0.59 </td> |
| − | <td>1.8</td> | + | <td align = "center"; valign = "center">1.8</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(C2)</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(C2)</p><strong></td> |
| − | <td>0.54 </td> | + | <td align = "center"; valign = "center">0.54 </td> |
| − | <td>1.6</td> | + | <td align = "center"; valign = "center">1.6</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
- Volumes are too large, so we aliquot the DNA gel slices in 2 x 2 ml Eppendorf for each tubes</br> | - Volumes are too large, so we aliquot the DNA gel slices in 2 x 2 ml Eppendorf for each tubes</br> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 25</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 817: | Line 822: | ||
</tr> | </tr> | ||
</thead> | </thead> | ||
| − | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(pET43.1a(+) 1 x2</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(pET43.1a(+) 1 x2</p><strong></td> |
| − | <td>225 </td> | + | <td align = "center"; valign = "center">225 </td> |
| − | <td>660</td> | + | <td align = "center"; valign = "center">660</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(pET43.1a(+) 2 x2</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(pET43.1a(+) 2 x2</p></strong></td> |
| − | <td>790</td> | + | <td align = "center"; valign = "center">790</td> |
| − | <td>900</td> | + | <td align = "center"; valign = "center">900</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(C1) x2</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(C1) x2</p><strong></td> |
| − | <td>295 </td> | + | <td align = "center"; valign = "center">295 </td> |
| − | <td>900</td> | + | <td align = "center"; valign = "center">900</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m(C2) x2</p><strong></td> | + | <td align = "center"; valign = "center"><strong><p>m(C2) x2</p><strong></td> |
| − | <td>270 </td> | + | <td align = "center"; valign = "center">270 </td> |
| − | <td>900</td> | + | <td align = "center"; valign = "center">900</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
| Line 861: | Line 864: | ||
- We have 4*20 µl tubes of pET43.1 so we regroup all of them in 1 tube. Finally, we have 1 tube for pET43.1 with 80 µl of DNA</br></br> | - We have 4*20 µl tubes of pET43.1 so we regroup all of them in 1 tube. Finally, we have 1 tube for pET43.1 with 80 µl of DNA</br></br> | ||
| − | + | <br /><br /><br /> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 877: | Line 878: | ||
<p> | <p> | ||
| − | <U> Aim:</U> We want to take back the plasmid and the inserts digested. </br> | + | <h6><U> Aim:</U></h6> We want to take back the plasmid and the inserts digested. </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></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | < | + | <h6><U>Method:</U></h6> |
| − | + | ||
Mixing all of reactants and let digest during 1h30 at 37 °C.</br> | Mixing all of reactants and let digest during 1h30 at 37 °C.</br> | ||
pET43.1 at 130 ng/µl will be digested by Hind III (NEB) and Xba I (NEB) | pET43.1 at 130 ng/µl will be digested by Hind III (NEB) and Xba I (NEB) | ||
| Line 889: | Line 889: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 26</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 900: | Line 901: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol DNA</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol DNA</p></strong></td> |
| − | <td>80 µl</td> | + | <td align = "center"; valign = "center">80 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol Hind III</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol Hind III</p></strong></td> |
| − | <td>15 µl</td> | + | <td align = "center"; valign = "center">15 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol Xba I</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol Xba I</p></strong></td> |
| − | <td>15 µl</td> | + | <td align = "center"; valign = "center">15 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol H<sub>2</sub>0</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol H<sub>2</sub>0</p></strong></td> |
| − | <td>0 µl</td> | + | <td align = "center"; valign = "center">0 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol buffer (10X) CutSmart</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol buffer (10X) CutSmart</p></strong></td> |
| − | <td>9 µl</td> | + | <td align = "center"; valign = "center">9 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol TOTAL</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol TOTAL</p></strong></td> |
| − | <td>93 µl</td> | + | <td align = "center"; valign = "center">93 µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | </br></br> | |
| − | + | ||
Let the digestion proceed for 1H and add an extra 0.5 µl of enzyme and continue the digestion for one hour more at 37 °C.</br> | Let the digestion proceed for 1H and add an extra 0.5 µl of enzyme and continue the digestion for one hour more at 37 °C.</br> | ||
| − | - Place the tubes in the freezer at -20 °C because it's late.</br> </br> | + | - Place the tubes in the freezer at -20 °C because it's late.</br></br> |
| − | + | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 952: | Line 952: | ||
<p> | <p> | ||
| − | <U> Aim:</U> Determine the quantity of DNA before doing the dephosphorylation</br> </br> | + | <h6><U> Aim:</U></h6> Determine the quantity of DNA before doing the dephosphorylation</br> </br> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | + | <h6><U>Method:</U></h6> | |
| − | + | ||
We use 1 µl of each sample and the Nanodrop</br> | We use 1 µl of each sample and the Nanodrop</br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 27</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 974: | Line 974: | ||
<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>10.4</td> | + | <td align = "center"; valign = "center">10.4</td> |
| − | <td>1.53</td> | + | <td align = "center"; valign = "center">1.53</td> |
| − | <td>0.20</td> | + | <td align = "center"; valign = "center">0.20</td> |
| − | <td>90.00</td> | + | <td align = "center"; valign = "center">90.00</td> |
| − | <td>900.00</td> | + | <td align = "center"; valign = "center">900.00</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C1</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C1</p></strong></td> |
| − | <td>9.00</td> | + | <td align = "center"; valign = "center">9.00</td> |
| − | <td>1.48</td> | + | <td align = "center"; valign = "center">1.48</td> |
| − | <td>0.04</td> | + | <td align = "center"; valign = "center">0.04</td> |
| − | <td>40.00</td> | + | <td align = "center"; valign = "center">40.00</td> |
| − | <td>360.00</td> | + | <td align = "center"; valign = "center">360.00</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C2</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C2</p></strong></td> |
| − | <td>5.70</td> | + | <td align = "center"; valign = "center">5.70</td> |
| − | <td>1.44</td> | + | <td align = "center"; valign = "center">1.44</td> |
| − | <td>0.10</td> | + | <td align = "center"; valign = "center">0.10</td> |
| − | <td>40.00</td> | + | <td align = "center"; valign = "center">40.00</td> |
| − | <td>240.00</td> | + | <td align = "center"; valign = "center">240.00</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | </br></br> | |
| − | + | ||
According to these results, we decided to reconcentrate the pET43.1 by using a PCR Clean-Up Kit from QIAGEN. We follow the kit steps.</br> | According to these results, we decided to reconcentrate the pET43.1 by using a PCR Clean-Up Kit from QIAGEN. We follow the kit steps.</br> | ||
| − | + |   • Spectrophotometer Ultrospec 3100 pro</br> | |
Dilute 1/50 in TE </br> | Dilute 1/50 in TE </br> | ||
A260<sub> nm</sub>=0.036</br> | A260<sub> nm</sub>=0.036</br> | ||
| Line 1,029: | Line 1,028: | ||
• Nanodrop: blank made with TE buffer</br> | • Nanodrop: blank made with TE buffer</br> | ||
| − | + | <table> | |
| + | <caption align="bottom" align="center"><i><p> <U>Table 28</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,045: | Line 1,045: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+)</br>digested on June 30</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+)</br>digested on June 30</p></strong></td> |
| − | <td>2.3</td> | + | <td align = "center"; valign = "center">2.3</td> |
| − | <td>0.360</td> | + | <td align = "center"; valign = "center">0.360</td> |
| − | <td>0.020</td> | + | <td align = "center"; valign = "center">0.020</td> |
| − | <td>0.009</td> | + | <td align = "center"; valign = "center">0.009</td> |
| − | <td>34.20</td> | + | <td align = "center"; valign = "center">34.20</td> |
| − | <td>1710.00</td> | + | <td align = "center"; valign = "center">1710.00</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>1.44</td> | + | <td align = "center"; valign = "center">1.44</td> |
| − | <td>0.58</td> | + | <td align = "center"; valign = "center">0.58</td> |
| − | <td>0.288</td> | + | <td align = "center"; valign = "center">0.288</td> |
| − | <td>0.259</td> | + | <td align = "center"; valign = "center">0.259</td> |
| − | <td>19.40</td> | + | <td align = "center"; valign = "center">19.40</td> |
| − | <td>2910.00</td> | + | <td align = "center"; valign = "center">2910.00</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C2</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C2</p></strong></td> |
| − | <td>1.49</td> | + | <td align = "center"; valign = "center">1.49</td> |
| − | <td>0.05</td> | + | <td align = "center"; valign = "center">0.05</td> |
| − | <td>0.229</td> | + | <td align = "center"; valign = "center">0.229</td> |
| − | <td>0.153</td> | + | <td align = "center"; valign = "center">0.153</td> |
| − | <td>11.40</td> | + | <td align = "center"; valign = "center">11.40</td> |
| − | <td>456.00</td> | + | <td align = "center"; valign = "center">456.00</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C1</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C1</p></strong></td> |
| − | <td>1.52</td> | + | <td align = "center"; valign = "center">1.52</td> |
| − | <td>0.13</td> | + | <td align = "center"; valign = "center">0.13</td> |
| − | <td>0.184</td> | + | <td align = "center"; valign = "center">0.184</td> |
| − | <td>0.121</td> | + | <td align = "center"; valign = "center">0.121</td> |
| − | <td>9.20</td> | + | <td align = "center"; valign = "center">9.20</td> |
| − | <td>368.00</td> | + | <td align = "center"; valign = "center">368.00</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | </br></br> | |
| − | + | ||
We pooled the two pET43.1a(+), we finally have 4620 ng of DNA in 200 µl.</br> | We pooled the two pET43.1a(+), we finally have 4620 ng of DNA in 200 µl.</br> | ||
| Line 1,094: | Line 1,093: | ||
Then, we measure again the quantity of DNA (blank made with NE)</br> | Then, we measure again the quantity of DNA (blank made with NE)</br> | ||
C = 29.5 ng/ µl so 590 ng of DNA in 20 µl.</br> | C = 29.5 ng/ µl so 590 ng of DNA in 20 µl.</br> | ||
| − | + | <br /><br /><br /> | |
| − | + | ||
</p> | </p> | ||
| Line 1,101: | Line 1,099: | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
<div class="lightbox" id="exp10"> | <div class="lightbox" id="exp10"> | ||
| Line 1,107: | Line 1,106: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Avoid the self religation of the digested plasmid.</br> This step makes the ligation with inserts easier, as the phosphate required for forming the phosphodiester bridge is provided by the insert, therefore the reaction occurs in trans.</br> </br> | + | <h6><U> Aim:</U></h6> Avoid the self religation of the digested plasmid.</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> | + | This step makes the ligation with inserts easier, as the phosphate required for forming the phosphodiester bridge is provided by the insert, therefore the reaction occurs in trans.</br> </br> |
| − | <U>What we did in the lab:</U></ | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/c0/T--Pasteur_Paris--dephosphorylation_protocol.pdf">link</a></br></br><br /> |
| − | + | <h6><U>What we did in the lab:</U></h6></br> | |
| − | <U>Method:</U></br> | + | |
| + | <h6><U>Method:</U></h6></br> | ||
1. In a 1 ml Eppendorf, we put 20 µl of DNA (590 ng of pET43.1)</br> | 1. In a 1 ml Eppendorf, we put 20 µl of DNA (590 ng of pET43.1)</br> | ||
1 µl of rSAP (recombinant shrimp alkaline phosphatase) (diluted 1/3), 2.4 µl of buffer and 0.6 µl of H<sub>2</sub>0</br> | 1 µl of rSAP (recombinant shrimp alkaline phosphatase) (diluted 1/3), 2.4 µl of buffer and 0.6 µl of H<sub>2</sub>0</br> | ||
2. Allow the digestion to proceed during 30 min at 37°C</br> | 2. Allow the digestion to proceed during 30 min at 37°C</br> | ||
3. Incubate 5 min at 65°C to inactivate the enzyme</br> | 3. Incubate 5 min at 65°C to inactivate the enzyme</br> | ||
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,129: | Line 1,129: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6> Make the plasmid bind with the inserts before the transformation. </br> </br> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a></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 /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method</U> | + | <h6><U>Method</U></h6> |
| − | 1. Put all reagents in 1 ml Eppendorf according to Table | + | 1. Put all reagents in 1 ml Eppendorf according to Table 29 </br> |
| − | + | ||
| + | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 29</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,149: | Line 1,150: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+)</br>dephosphorylated (100 ng)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+)</br>dephosphorylated (100 ng)</p></strong></td> |
| − | <td>3.5</td> | + | <td align = "center"; valign = "center">3.5</td> |
| − | <td>3.5</td> | + | <td align = "center"; valign = "center">3.5</td> |
| − | <td>0.0</td> | + | <td align = "center"; valign = "center">0.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C1/C2 </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C1/C2 </p></strong></td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>0.0</td> | + | <td align = "center"; valign = "center">0.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>T4 DNA ligase</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>T4 DNA ligase</p></strong></td> |
| − | <td>1.0</td> | + | <td align = "center"; valign = "center">1.0</td> |
| − | <td>1.0</td> | + | <td align = "center"; valign = "center">1.0</td> |
| − | <td>1.0</td> | + | <td align = "center"; valign = "center">1.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>buffer 10X</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>buffer 10X</p></strong></td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | < | + | <br /></br> |
| − | + | <font color = "red">   &9888; Be careful! We have 6 tubes: 3 for C1 insert and 3 for C2</font></br> | |
2. Let ligation proceed for 10 min at room temperature</br> | 2. Let ligation proceed for 10 min at room temperature</br> | ||
3. Incubate for 10 min at 65°C</br> | 3. Incubate for 10 min at 65°C</br> | ||
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,198: | Line 1,199: | ||
<p> | <p> | ||
| − | <U> Aim:</U> Recover the ligation products, amplify them.</br>.</br> | + | <h6><U> Aim:</U></h6> Recover the ligation products, amplify them.</br>.</br> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_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>Cloning efficiency competent DH5-α (10<sup>6</sup>) | + | <h6><U>Material</U></h6> |
| − | <U>Method:</U></ | + | • Cloning efficiency competent DH5-&alpha <br;(10<sup>6</sup>)<br /> |
| + | • SOC media <br /> | ||
| + | • 42 °C water bath <br /><br /> | ||
| + | <h6><U>Method:</U></h6> | ||
We aliquot the competent cells in 5 tubes (80 µl per tube) and we use all our products of ligation. (refer to June 6, 2016 for detailed protocol)</br> | We aliquot the competent cells in 5 tubes (80 µl per tube) and we use all our products of ligation. (refer to June 6, 2016 for detailed protocol)</br> | ||
| − | The cells were incubated on ice for 30 | + | The cells were incubated on ice for 30 minutes. The heat shocked for 40 secs at 42°C. They were the placed on ice for 3 min. We added 150 µl of SOC media.</br> |
We placed our tubes in the shaking incubator at 37°C and 130 RPM during 40 min. Then, we spread on petri dishes using plastic rakes, and incubated at 37°C during the night.</br></br></br> | We placed our tubes in the shaking incubator at 37°C and 130 RPM during 40 min. Then, we spread on petri dishes using plastic rakes, and incubated at 37°C during the night.</br></br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 30</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| − | <th | + | <th>λ= 260<sub>nm</sub> </th> |
<th>C1</th> | <th>C1</th> | ||
<th>C2</th> | <th>C2</th> | ||
| Line 1,218: | Line 1,223: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260 nm</span> /A<span>280 nm</span> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<span>260 nm</span> /A<span>280 nm</span> </p></strong></td> |
| − | <td>1.71</td> | + | <td align = "center"; valign = "center">1.71</td> |
| − | <td>1.80</td> | + | <td align = "center"; valign = "center">1.80</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260 nm</span> /A<span>230 nm</span> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<span>260 nm</span> /A<span>230 nm</span> </p></strong></td> |
| − | <td>0.39</td> | + | <td align = "center"; valign = "center">0.39</td> |
| − | <td>0.39</td> | + | <td align = "center"; valign = "center">0.39</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinal </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinal </p></strong></td> |
| − | <td>9.7 ng/µl</td> | + | <td align = "center"; valign = "center">9.7 ng/µl</td> |
| − | <td> 11.0 ng/µl </td> | + | <td align = "center"; valign = "center"> 11.0 ng/µl </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table></br></br> |
| − | + | <br /><br /><br /> | |
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,251: | Line 1,255: | ||
<p> | <p> | ||
| − | <U> Aim:</U> To separate the digested DNA from undigested DNA. </br> | + | <h6><U> Aim:</U></h6> To separate the digested DNA from undigested DNA. </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a></br></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>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> |
•pET43.1a plasmid (obtained with Midiprep done June 16, 2016)</br> | •pET43.1a plasmid (obtained with Midiprep done June 16, 2016)</br> | ||
•pET43.1a plasmid cutted by and extracted from gel 29/06/16 and digested again by same enzymes</br> | •pET43.1a plasmid cutted by and extracted from gel 29/06/16 and digested again by same enzymes</br> | ||
| Line 1,263: | Line 1,267: | ||
•P10 pipet, P20 pipet, test tube 250mL, electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator, centrifuge 5415D. </br></br> | •P10 pipet, P20 pipet, test tube 250mL, electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator, centrifuge 5415D. </br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. Put the gel in the electrophoresis chamber and add TAE 0.5X buffer solution to cover the gel.</br> | 1. Put the gel in the electrophoresis chamber and add TAE 0.5X buffer solution to cover the gel.</br> | ||
2. Following this deposit table:</br> | 2. Following this deposit table:</br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 31</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,280: | Line 1,285: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Name</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Name</p></strong></td> |
| − | <td>Marker </br> weight</td> | + | <td align = "center"; valign = "center">Marker </br> weight</td> |
| − | <td>pET43.1a(+) </br>uncut</td> | + | <td align = "center"; valign = "center">pET43.1a(+) </br>uncut</td> |
| − | <td>pET43.1a(+) </br> X/B</td> | + | <td align = "center"; valign = "center">pET43.1a(+) </br> X/B</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA μl </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA μl </p></strong></td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">50</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">50</td> |
| − | <td>50</td> | + | <td align = "center"; valign = "center">50</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>H<span>2</span> 0 µl</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>H<span>2</span> 0 µl</p></strong></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>loading buffer 6X µl</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>loading buffer 6X µl</p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
Run at 90 V.</br></br> | Run at 90 V.</br></br> | ||
| Line 1,323: | Line 1,327: | ||
- Top of agarose gel--> 6000 bp</br> | - Top of agarose gel--> 6000 bp</br> | ||
| − | <U>Results:</U></ | + | <h6><U>Results:</U></h6> |
Agarose gel:</br> | Agarose gel:</br> | ||
| − | We can see on this second gel, pET43.1 cut there are 2 bands (the first is pET43.1 digested half, the second band is pET43.1 totally digested) again. We left the migration more longer to separate better the plasmid totally digested from the half digested one.</br> | + | We can see on this second gel, pET43.1 cut there are 2 bands (the first is pET43.1 digested half, the second band is pET43.1 totally digested) again. We left the migration more longer to separate better the plasmid totally digested from the half digested one.</br><br /> |
| − | + | ||
| − | < | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | <center><img src="https://static.igem.org/mediawiki/2016/5/5d/5._fig5_Pasteur.png " width="300px"; alt=""><i><p><U>Figure 5</U></p></i></center></br></br> | ||
| + | <center><img src=" https://static.igem.org/mediawiki/2016/3/31/6.fig6_Pasteur.png" width="300px"; alt=""></center><i><p><U>Figure 6</U></p></i></center></br></br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,348: | Line 1,349: | ||
<p> | <p> | ||
| − | <U> Aim:</U> </br></br> | + | <h6><U> Aim:</U></h6> </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4e/T--Pasteur_Paris--Gel_extraction_protocol.pdf">link</a></br></br> | + | <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>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. Cut agarose gel to have only the digested plasmid that we need and put it on a 2 ml Eppendorf.</br> | 1. Cut agarose gel to have only the digested plasmid that we need and put it on a 2 ml Eppendorf.</br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 32</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,367: | Line 1,369: | ||
<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>520</td> | + | <td align = "center"; valign = "center">520</td> |
| − | <td>1.073 </br>uncut</td> | + | <td align = "center"; valign = "center">1.073 </br>uncut</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
2. Used PCR clean-up Gel Extraction Kit (Macherey-Nagel)</br> | 2. Used PCR clean-up Gel Extraction Kit (Macherey-Nagel)</br> | ||
| Line 1,389: | Line 1,390: | ||
Elute DNA with 3 x 15 µl/Eppendorf-> final volume = 45 µl of DNA.</br> | Elute DNA with 3 x 15 µl/Eppendorf-> final volume = 45 µl of DNA.</br> | ||
| − | </br> | + | </br><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,402: | Line 1,403: | ||
<p> | <p> | ||
| − | <U> Aim:</U>We had problems to measure our DNA concentration with our spectrophotometer so we use a Nanodrop (Thermofisher) </br></br> | + | <h6><U> Aim:</U></h6> We had problems to measure our DNA concentration with our spectrophotometer so we use a Nanodrop (Thermofisher) </br></br> |
| − | <U>Results:</U></br> | + | <h6><U>Results:</U></h6></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 33</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,418: | Line 1,420: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A260<sub>nm</sub>/A280<sub>nm</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A260<sub>nm</sub>/A280<sub>nm</sub></p></strong></td> |
| − | <td>1.48</td> | + | <td align = "center"; valign = "center">1.48</td> |
| − | <td>1.44 </td> | + | <td align = "center"; valign = "center">1.44 </td> |
| − | <td>1.53 </td> | + | <td align = "center"; valign = "center">1.53 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A260<sub>nm</sub>/A230<sub>nm</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A260<sub>nm</sub>/A230<sub>nm</sub></p></strong></td> |
| − | <td>0.04</td> | + | <td align = "center"; valign = "center">0.04</td> |
| − | <td>0.10 </td> | + | <td align = "center"; valign = "center">0.10 </td> |
| − | <td>0.20 </td> | + | <td align = "center"; valign = "center">0.20 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinal (ng/µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinal (ng/µl)</p></strong></td> |
| − | <td>9.0 </td> | + | <td align = "center"; valign = "center">9.0 </td> |
| − | <td>5.7 </td> | + | <td align = "center"; valign = "center">5.7 </td> |
| − | <td>10.4 </td> | + | <td align = "center"; valign = "center">10.4 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
• DNA reconcentration:</br> | • DNA reconcentration:</br> | ||
| − | To dephosphorylate our vector, we need use less than 50 µ | + | To dephosphorylate our vector, we need use less than 50 µl, so we reconcentrate this with the PCR clean-up Gel Extraction Kit (Macherey-Nagel).</br> |
NTI volumes: </br> | NTI volumes: </br> | ||
| Line 1,460: | Line 1,461: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 34</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,473: | Line 1,475: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<sub>260 nm</sub>/A<sub>280 nm</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<sub>260 nm</sub>/A<sub>280 nm</sub></p></strong></td> |
| − | <td>1.49</td> | + | <td align = "center"; valign = "center">1.49</td> |
| − | <td>1.44 </td> | + | <td align = "center"; valign = "center">1.44 </td> |
| − | <td>1.53 </td> | + | <td align = "center"; valign = "center">1.53 </td> |
| − | <td>1.44 </td> | + | <td align = "center"; valign = "center">1.44 </td> |
| − | <td>1.89 </td> | + | <td align = "center"; valign = "center">1.89 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A260<sub> nm</sub>/A230<sub> nm</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A260<sub> nm</sub>/A230<sub> nm</sub></p></strong></td> |
| − | <td>0.05</td> | + | <td align = "center"; valign = "center">0.05</td> |
| − | <td>0/10 </td> | + | <td align = "center"; valign = "center">0/10 </td> |
| − | <td>0.20 </td> | + | <td align = "center"; valign = "center">0.20 </td> |
| − | <td>0.58 </td> | + | <td align = "center"; valign = "center">0.58 </td> |
| − | <td>1.89 </td> | + | <td align = "center"; valign = "center">1.89 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinal (ng/µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinal (ng/µl)</p></strong></td> |
| − | <td>11.4</td> | + | <td align = "center"; valign = "center">11.4</td> |
| − | <td>9.2 </td> | + | <td align = "center"; valign = "center">9.2 </td> |
| − | <td>10.4 </td> | + | <td align = "center"; valign = "center">10.4 </td> |
| − | <td>19.4 </td> | + | <td align = "center"; valign = "center">19.4 </td> |
| − | <td>94.4 </td> | + | <td align = "center"; valign = "center">94.4 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA quantity ng</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA quantity ng</p></strong></td> |
| − | <td>456</td> | + | <td align = "center"; valign = "center">456</td> |
| − | <td>368 </td> | + | <td align = "center"; valign = "center">368 </td> |
| − | <td>1710 </td> | + | <td align = "center"; valign = "center">1710 </td> |
| − | <td>2910 </td> | + | <td align = "center"; valign = "center">2910 </td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
So we regrouped 2 tubes of pET43.1a digested solution in one. We had 4620 ng DNA in 200 µl and we tried to reconcentrate it with the PCR clean-up Gel extraction Kit (Macherey-Nagel) in 20 µl.</br> | So we regrouped 2 tubes of pET43.1a digested solution in one. We had 4620 ng DNA in 200 µl and we tried to reconcentrate it with the PCR clean-up Gel extraction Kit (Macherey-Nagel) in 20 µl.</br> | ||
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,525: | Line 1,526: | ||
<p> | <p> | ||
| − | <U>Results:</U></br> | + | <h6><U>Results:</U></h6><br /> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 35</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,538: | Line 1,540: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A260<sub> nm</sub>/A280<sub> nm</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A260<sub> nm</sub>/A280<sub> nm</sub></p></strong></td> |
| − | <td>1.49</td> | + | <td align = "center"; valign = "center">1.49</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A260<sub> nm</sub>/A230<sub> nm</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A260<sub> nm</sub>/A230<sub> nm</sub></p></strong></td> |
| − | <td>0.35</td> | + | <td align = "center"; valign = "center">0.35</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinal (ng/µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinal (ng/µl)</p></strong></td> |
| − | <td>29.5 </td> | + | <td align = "center"; valign = "center">29.5 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA quantity</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA quantity</p></strong></td> |
| − | <td>520 ng </td> | + | <td align = "center"; valign = "center">520 ng </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
• DNA reconcentration:</br> | • DNA reconcentration:</br> | ||
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
<div class="lightbox" id="exp17"> | <div class="lightbox" id="exp17"> | ||
| Line 1,573: | Line 1,575: | ||
<p> | <p> | ||
| − | <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> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/c0/T--Pasteur_Paris--dephosphorylation_protocol.pdf">link</a></br></br><br /> |
| − | <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. Desphosphorylation:</br> | 1. Desphosphorylation:</br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 36</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,588: | Line 1,591: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol DNA(µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol DNA(µl)</p></strong></td> |
| − | <td>20</td> | + | <td align = "center"; valign = "center">20</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol rSAP diluted 1/3 (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol rSAP diluted 1/3 (µl)</p></strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol H<sub>2</sub>0 (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol H<sub>2</sub>0 (µl)</p></strong></td> |
| − | <td>0.6 </td> | + | <td align = "center"; valign = "center">0.6 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol Buffer (10X) Cutsmart (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol Buffer (10X) Cutsmart (µl)</p></strong></td> |
| − | <td>2.4 </td> | + | <td align = "center"; valign = "center">2.4 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol Total (µl) </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol Total (µl) </p></strong></td> |
| − | <td>24 </td> | + | <td align = "center"; valign = "center">24 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
• DNA reconcentration:</br> | • DNA reconcentration:</br> | ||
| Line 1,627: | Line 1,629: | ||
2. Ligation:</br> | 2. Ligation:</br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 37</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,638: | Line 1,641: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><p>pET43.1a(+) (50 mg)</p></td> | + | <td align = "center"; valign = "center"><p>pET43.1a(+) (50 mg)</p></td> |
| − | <td>3.5</td> | + | <td align = "center"; valign = "center">3.5</td> |
| − | <td>3.5</td> | + | <td align = "center"; valign = "center">3.5</td> |
| − | <td>3.5</td> | + | <td align = "center"; valign = "center">3.5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><p>Insert (C1/C2)</p></td> | + | <td align = "center"; valign = "center"><p>Insert (C1/C2)</p></td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>0.0</td> | + | <td align = "center"; valign = "center">0.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><p>T4 Ligase</p></td> | + | <td align = "center"; valign = "center"><p>T4 Ligase</p></td> |
| − | <td>1.0</td> | + | <td align = "center"; valign = "center">1.0</td> |
| − | <td>1.0</td> | + | <td align = "center"; valign = "center">1.0</td> |
| − | <td>1.0</td> | + | <td align = "center"; valign = "center">1.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><p>Buffer 10X</p></td> | + | <td align = "center"; valign = "center"><p>Buffer 10X</p></td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><p>H<sub>2</sub>0</p></td> | <td><p>H<sub>2</sub>0</p></td> | ||
| − | <td>7.5</td> | + | <td align = "center"; valign = "center">7.5</td> |
| − | <td>4.0</td> | + | <td align = "center"; valign = "center">4.0</td> |
| − | <td>9.0</td> | + | <td align = "center"; valign = "center">9.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><p>TOTAL</p></td> | + | <td align = "center"; valign = "center"><p>TOTAL</p></td> |
| − | <td>15.0</td> | + | <td align = "center"; valign = "center">15.0</td> |
| − | <td>15.0</td> | + | <td align = "center"; valign = "center">15.0</td> |
| − | <td>15.0</td> | + | <td align = "center"; valign = "center">15.0</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
Incubate 10 min at room temperature</br> | Incubate 10 min at room temperature</br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,692: | Line 1,695: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,704: | Line 1,708: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U>Aim:</U>After this third transformation without colony, we did an electrophoresis with ligation products. It is also oo check if the ligation was successful or not. This is unusual as the amount of material used is quite low. However, without proof of the ligation working we cannot ascertain many parameters, including transformaton procedures, presence of DNA, etc..</br></br> | + | <h6><U>Aim:</U><h/h6> After this third transformation without colony, we did an electrophoresis with ligation products. It is also oo check if the ligation was successful or not. This is unusual as the amount of material used is quite low. However, without proof of the ligation working we cannot ascertain many parameters, including transformaton procedures, presence of DNA, etc..</br></br> |
| − | <U> Protocol:</U> follow in this link<a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br> | + | <h6><U> Protocol:</U></h6> follow in this link<a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br><br /> |
| − | <U> What we did on the lab:</U></br> | + | <h6><U> What we did on the lab:</U></h6></br> |
| − | <U> Material:</U></ | + | <h6><U> Material:</U></h6> |
• pET43.1a plasmid (obtained with midiprep done 16/06/16)</br> | • pET43.1a plasmid (obtained with midiprep done 16/06/16)</br> | ||
• pET43.1a plasmid cut by Hind III/Xba I</br> | • pET43.1a plasmid cut by Hind III/Xba I</br> | ||
| Line 1,718: | Line 1,722: | ||
• P10 pipet, P20 pipet, test tube 250 ml,</br> electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge 5415D, </br></br> | • P10 pipet, P20 pipet, test tube 250 ml,</br> electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge 5415D, </br></br> | ||
| − | <U> Method:</U></ | + | <h6><U> Method:</U></h6> |
1. We redo the ligation of pET43.1 digested with C1 (refer on the June 30, 2016 for protocol) but we let the ligation run for one hour at RT.</br> | 1. We redo the ligation of pET43.1 digested with C1 (refer on the June 30, 2016 for protocol) but we let the ligation run for one hour at RT.</br> | ||
2. We made a 0.7% agarose gel</br> | 2. We made a 0.7% agarose gel</br> | ||
| Line 1,725: | Line 1,729: | ||
Deposit table :</br> | Deposit table :</br> | ||
| − | L1 --> Ladder 10 | + | L1 --> Ladder 10 µl (Gene ruler)</br> |
L2-4 -->pET43.1 uncut (reference at 7000 bp)</br> | L2-4 -->pET43.1 uncut (reference at 7000 bp)</br> | ||
L5-7 --> pET43.1 digested and dephosphorylated</br> | L5-7 --> pET43.1 digested and dephosphorylated</br> | ||
| Line 1,734: | Line 1,738: | ||
<U>Results:</U>We see nothing on the gel, no DNA in our tubes. We probably made a mistake when we performed the experiment.</br> | <U>Results:</U>We see nothing on the gel, no DNA in our tubes. We probably made a mistake when we performed the experiment.</br> | ||
| − | + | <br /><br /><br /> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,743: | Line 1,745: | ||
| − | |||
| − | |||
</body> | </body> | ||
</html> | </html> | ||
