Difference between revisions of "Team:Pasteur Paris/Microbiology week2"
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<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> | ||
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<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> |
| − | <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> | + | 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 /> |
| + | <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> | ||
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</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> | ||
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</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table></br> |
| − | </br> | + | </br><br /><br /> |
| − | < | + | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<a href="# exp2" class="closemsg"></a> | <a href="# exp2" class="closemsg"></a> | ||
<figcaption><p> | <figcaption><p> | ||
| − | <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> |
• 1X TAE buffer </br> | • 1X TAE buffer </br> | ||
• Agarose </br> | • Agarose </br> | ||
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• 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> | ||
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<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> | ||
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<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 5</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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</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> | ||
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<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> | ||
| − | |||
| − | |||
| − | |||
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<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://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a></br></br> | + | <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> | ||
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<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://static.igem.org/mediawiki/2016/b/b1/Stab_culture_Pasteur_Paris_2016.pdf">link</a></br></br> | + | 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> | ||
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&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> | ||
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</tbody> | </tbody> | ||
</table> | </table> | ||
| − | </br> | + | </br><br /><br /> |
| − | < | + | |
| − | < | + | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
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</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
| − | |||
</div> | </div> | ||
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<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> | ||
Revision as of 20:13, 19 October 2016
