Difference between revisions of "Team:Pasteur Paris/Microbiology week2"
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{{Paris_Pasteur_Microbiology}} | {{Paris_Pasteur_Microbiology}} | ||
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<a href="#exp10"><h4> 14. Dephosphorylation </h4></a></br> | <a href="#exp10"><h4> 14. Dephosphorylation </h4></a></br> | ||
<a href="#exp11"><h4> 15. Ligation of C1 and C2 with pET43.1a(+) </h4></a></br> | <a href="#exp11"><h4> 15. Ligation of C1 and C2 with pET43.1a(+) </h4></a></br> | ||
| + | </p> | ||
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<div class="lightbox" id="exp1"> | <div class="lightbox" id="exp1"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp1" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.</br> Seed pET43.1 DH5α in LB + carbenicillin 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 combinations need to be stored beyond the lifetime of plate colonies. </br></br> | + | <U> Aim:</U> 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> |
| − | <U> Protocol:</U> follow in this | + | <U> Protocol:</U> follow in this link</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 & | + | <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> |
<table> | <table> | ||
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<div class="lightbox" id="exp2"> | <div class="lightbox" id="exp2"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp2" class="closemsg"></a> |
<figcaption><p> | <figcaption><p> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
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</br> | </br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | 1. Buffer solution: we have 1.0X stock TAE solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer </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> |
| − | 2. Agarose gel: We prepared 0.7% w/v agarose | + | 2. Agarose gel: We prepared 0.7% w/v agarose in 1X TAE and allow it to set.</br></br> |
<B> For agarose gel electrophoresis experiment:</B></br></br> | <B> For agarose gel electrophoresis experiment:</B></br></br> | ||
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• 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 | + | • pET43.1a plasmid digested by BamH I/Hind III</br> |
• gel 0.7% agarose</br> | • gel 0.7% agarose</br> | ||
• TAE 0.5X buffer</br> | • TAE 0.5X buffer</br> | ||
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The digestion has worked since the plasmids digested have moved faster. </br></br>NB: the gel suffered from overheating. </br> | The digestion has worked since the plasmids digested have moved faster. </br></br>NB: the gel suffered from overheating. </br> | ||
</br></br></br></br> | </br></br></br></br> | ||
| − | + | Figure 1: </br></br> | |
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| − | + | ||
| − | + | ||
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<div class="lightbox" id="exp3"> | <div class="lightbox" id="exp3"> | ||
<figure> | <figure> | ||
| − | <a href="#" 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> | <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> | ||
| − | <U> Protocol:</U> follow in this | + | <U> Protocol:</U> follow in this link</br></br> |
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U> | <U>Materials:</U> | ||
• 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> |
• Shaking incubator (INFORS HT)</br> | • Shaking incubator (INFORS HT)</br> | ||
| − | • Antibiotic: | + | • 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> | ||
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<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | 1. Pick one colony and put it into 5 ml of LB medium + 2 | + | 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> | ||
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<div class="lightbox" id="exp4"> | <div class="lightbox" id="exp4"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp4" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
<U> Aim:</U> Use the preculture of June 13, 2016 to start the new culture.</br></br> | <U> Aim:</U> Use the preculture of June 13, 2016 to start the new culture.</br></br> | ||
| − | <U> Protocol:</U> follow in this | + | <U> Protocol:</U> follow in this link</br></br> |
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
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<U>Method:</U></br> | <U>Method:</U></br> | ||
1. Get back preculture performed on the June 13, 2016, put it in the swing bucket centrifuge during 7 min (3500 RPM) and remove the supernatant. </br> | 1. Get back preculture performed on the June 13, 2016, put it in the swing bucket centrifuge during 7 min (3500 RPM) and remove the supernatant. </br> | ||
| − | 2. Add 5ml of fresh | + | 2. Add 5ml of fresh LB, resuspend the pellet and centrifuge again at 3500 RPM during 7 min. </br> |
3. Repeat one more time step 2</br> | 3. Repeat one more time step 2</br> | ||
| − | 4. Resuspend the pellet into 5 mL of fresh LB+ 2.5 | + | 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 RPM) Overnight</br> | 5. Grow in the shaking incubator (T= 37 °C / 130 RPM) Overnight</br> | ||
</br></br> | </br></br> | ||
| − | We overshot the 0.7 OD, | + | We overshot the 0.7 OD, set point therefore we continued overnight. </br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<div class="lightbox" id="exp5"> | <div class="lightbox" id="exp5"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp5" class="closemsg"></a> |
<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 nm ( | + | <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> |
| − | <U> Protocol:</U> follow in this | + | <U> Protocol:</U> follow in this link</br></br> |
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| − | • Falcon of | + | • Falcon of 50 ml </br> |
• LB autoclaved 500 ml<br> | • LB autoclaved 500 ml<br> | ||
| − | • | + | • LB (Luria Broth) autoclaved (500 ml) </br> |
&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> | ||
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</br> | </br> | ||
<U>Method:</U></br> 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> | <U>Method:</U></br> 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> | ||
| − | 1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at | + | 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 min onwards. We recorded OD600<sub>nm</sub> for this specific experiment after 3 hours.</br> |
</br></br> | </br></br> | ||
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<U>Growth curve :</U> | <U>Growth curve :</U> | ||
| − | + | </br> Figure 2: Growth curve for DH5α-pET43.1a(+) by measurement of OD600<sub>nm</sub>. O-time point corresponds to 12h37</br> | |
| − | + | ||
</p> | </p> | ||
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<div class="lightbox" id="exp6"> | <div class="lightbox" id="exp6"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp6" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
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<U> What we did on the lab:</U></br> | <U> What we did on the lab:</U></br> | ||
<U> Method:</U></br> | <U> Method:</U></br> | ||
| − | 1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 l 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> | ||
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<div class="lightbox" id="exp7"> | <div class="lightbox" id="exp7"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp7" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> we received our gene block | + | <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> 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> | ||
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<th>Name </th> | <th>Name </th> | ||
<th>Weight (mg)</th> | <th>Weight (mg)</th> | ||
| − | <th>Cfinal (ng/µ ;l)</th> | + | <th>Cfinal (ng/µl)</th> |
| − | <th>V(TE) (µ ;l)</th> | + | <th>V(TE) (µl)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
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<div class="lightbox" id="exp8"> | <div class="lightbox" id="exp8"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp8" class="closemsg"></a> |
<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. | + | <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 |
</br></br> | </br></br> | ||
| − | <U> Protocol:</U> follow in this | + | <U> Protocol:</U> follow in this link</br></br> |
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Material:</U></br> | <U>Material:</U></br> | ||
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</br> | </br> | ||
<center>Table 8</center></br> </br> | <center>Table 8</center></br> </br> | ||
| − | 3. Incubate the samples between 37-55°C vortexing periodically until the gel slice is completely dissolved (/ ~ | + | 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 min then discard flow | + | 4. Load sample onto the column. Close the cap, spin for 1 min then discard flow through</br> |
| − | 5. Re insert column into collection tube. Add 200 µl 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 | + | 8. Add 6 µl of DNA Elution buffer to the center of the matric. Wait for 1 min and spin for 1 min to elute DNA</br> |
</p> | </p> | ||
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<div class="lightbox" id="exp9"> | <div class="lightbox" id="exp9"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp9" class="closemsg"></a> |
<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> | <U> Aim:</U> 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 | + | <U> Protocol:</U> follow in this link</br></br> |
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Material:</U></br></br> | <U>Material:</U></br></br> | ||
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Add all reagents in 1 ml Eppendorf </br></br> | Add all reagents in 1 ml Eppendorf </br></br> | ||
- Digest during 2 h at 37 °C </br> | - Digest during 2 h at 37 °C </br> | ||
| − | - For the reagent volumes, refer to the Table 9. Total volume = 50 | + | - For the reagent volumes, refer to the Table 9. Total volume = 50 µl</br></br> |
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</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> | + | <td><strong><p>BamH I (µl)</p></strong></td> |
<td>1</td> | <td>1</td> | ||
<td>1</td> | <td>1</td> | ||
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<tr> | <tr> | ||
| − | <td><strong><p> | + | <td><strong><p>Hind III (µl)</ </p></strong></td> |
<td>2</td> | <td>2</td> | ||
<td>2</td | <td>2</td | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> | + | <td><strong><p> H<sub>2</sub>0 (µl)</ </p></strong></td> |
<td>22</td> | <td>22</td> | ||
<td>22</td> | <td>22</td> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U | + | <U> Aim:</U</br></br> |
| − | <U> Protocol:</U> follow in this | + | <U> Protocol:</U> follow in this link</br></br> |
</p> | </p> | ||
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<div class="lightbox" id="exp11"> | <div class="lightbox" id="exp11"> | ||
<figure> | <figure> | ||
| − | <a href="#" class="closemsg"></a> | + | <a href="# exp11" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U | + | <U> Aim:</U</br></br> |
| − | <U> Protocol:</U> follow in this | + | <U> Protocol:</U> follow in this link</br></br> |
</p> | </p> | ||
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</div> | </div> | ||
</div> | </div> | ||
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</body> | </body> | ||
| − | + | <html> | |
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Revision as of 20:43, 9 October 2016
