Difference between revisions of "Team:Pasteur Paris/Microbiology week1"
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<center></a><a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><img src="https://static.igem.org/mediawiki/2016/5/5a/Labwork_pasteur.png" width="40%" alt=""/></img></a></center> | <center></a><a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><img src="https://static.igem.org/mediawiki/2016/5/5a/Labwork_pasteur.png" width="40%" alt=""/></img></a></center> | ||
</div> | </div> | ||
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<div id="week1"> | <div id="week1"> | ||
<p><h5><B>Week 1</B></h5></p> | <p><h5><B>Week 1</B></h5></p> | ||
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<a href="#exp4"><h4> 4. Digestion of the plasmid pSB1C3 and pET43.1a(+) </h4></a></br> | <a href="#exp4"><h4> 4. Digestion of the plasmid pSB1C3 and pET43.1a(+) </h4></a></br> | ||
</p> | </p> | ||
| + | </div> | ||
<div class="lightbox" id="exp1"> | <div class="lightbox" id="exp1"> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>To increase the amount of plasmid by transformation in competent cells.< | + | <h6><U> Aim :</U></h6>To increase the amount of plasmid by transformation in competent cells.<br />The amount of plasmid supplied is insufficient to perform all of our future experiments. Therefore we need to amplify the amount of plasmids. </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> |
| − | <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> |
• subcloning competent cells</br> | • subcloning competent cells</br> | ||
• pSB1C3 plasmid (from shipped BioBrick-competent cells testing kit), chloramphenicol resistance</br> | • pSB1C3 plasmid (from shipped BioBrick-competent cells testing kit), chloramphenicol resistance</br> | ||
| Line 260: | Line 273: | ||
• SOC (Super optimal broth) media</br> | • SOC (Super optimal broth) media</br> | ||
• LB (Luria broth) Agar plates containing 50 µg/ml carbenicillin or 34 µg/ml chloramphenicol</br> | • LB (Luria broth) Agar plates containing 50 µg/ml carbenicillin or 34 µg/ml chloramphenicol</br> | ||
| − | • Microbiology equipment ( | + | • 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> |
| − | <U>Method:</U></ | + | <h6><U>Method :</U></h6> |
1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells in volume of 50 µl/vial.</br> | 1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells in volume of 50 µl/vial.</br> | ||
2. Add 50 pg plasmids to each 50 µl of competent cells vial and tap gently.</br> | 2. Add 50 pg plasmids to each 50 µl of competent cells vial and tap gently.</br> | ||
| − | 3. Place on ice for 30 | + | 3. Place on ice for 30 minutes. Meanwhile, warm LB agar plates from cold room in 37°C non shaking incubator.</br> |
| − | 4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 | + | 4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 minutes.</br> |
5. Add 500 µl of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).</br> | 5. Add 500 µl of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).</br> | ||
6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.</br> | 6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.</br> | ||
7. Near Bunsen burner flame, add 45 µl of competent cells +135 µl of SOC or 200 µl aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.</br> | 7. Near Bunsen burner flame, add 45 µl of competent cells +135 µl of SOC or 200 µl aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.</br> | ||
8. Place plates inverted in the static 37°C incubator overnight. </br> | 8. Place plates inverted in the static 37°C incubator overnight. </br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
<div class="lightbox" id="exp2"> | <div class="lightbox" id="exp2"> | ||
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<a href="# exp2" class="closemsg"></a> | <a href="# exp2" class="closemsg"></a> | ||
<figcaption><p> | <figcaption><p> | ||
| − | <U> Aim:</U> To start a culture for Miniprep. </br>In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.</br></br> | + | <h6><U> Aim :</U></h6> To start a culture for Miniprep. </br>In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.</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> |
| − | <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> |
| − | • Microbiology | + | • 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> |
• 25 ml flasks<br> | • 25 ml flasks<br> | ||
• Carbenicillin 50 mg/ml</br> | • Carbenicillin 50 mg/ml</br> | ||
• Chloramphenicol 34 mg/ml</br> | • Chloramphenicol 34 mg/ml</br> | ||
• LB medium</br></br> | • LB medium</br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1.One colony is picked from the plates and shaken in 25 ml of LB supplemented with carbenicillin or chloramphenicol at 50 mg/ml or 34 mg/ml respectively. </br> | 1.One colony is picked from the plates and shaken in 25 ml of LB supplemented with carbenicillin or chloramphenicol at 50 mg/ml or 34 mg/ml respectively. </br> | ||
2.The flask is placed in a shaking incubator at 37°C, 150 rpm overnight. </br> | 2.The flask is placed in a shaking incubator at 37°C, 150 rpm overnight. </br> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
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| + | |||
<div class="lightbox" id="exp3"> | <div class="lightbox" id="exp3"> | ||
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<a href="# exp3" class="closemsg"></a> | <a href="# exp3" class="closemsg"></a> | ||
<figcaption><p> | <figcaption><p> | ||
| − | <U> Aim:</U> To perform a midiprep to isolate plasmid DNA of pSB1C3 and pET43.1a(+) </br> | + | <h6><U> Aim:</U></h6> To perform a midiprep to isolate plasmid DNA of pSB1C3 and pET43.1a(+) </br> |
The amplification method to increase the amount of plasmid is called Mini or Midiprep.</br></br> | The amplification method to increase the amount of plasmid is called Mini or Midiprep.</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> |
| − | <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> |
• 50 ml Falcon tube</br> | • 50 ml Falcon tube</br> | ||
• Shaking incubator (INFORS HT)<br> | • Shaking incubator (INFORS HT)<br> | ||
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• Swing bucket centrifuge (JOUAN GR41)</br> | • Swing bucket centrifuge (JOUAN GR41)</br> | ||
• QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)</br></br> | • QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)</br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6>The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 20 minutes. We will follow most of the protocol of QIAGEN Midiprep 2016 except for a few modifications, which we describe, therefore, below. </br><br /> |
| − | 1. Use culture from overnight (17 | + | 1. Use culture from overnight (17 hours) step on June 7, 2016. </br> |
| − | 2. Pour culture in 50 ml Falcon and centrifuge (15 min, 3500g, 4°C) </br> | + | 2. Pour culture in 50 ml Falcon and centrifuge (15 min, 3500g, 4°C). </br> |
| − | 3. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet </br> | + | 3. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet. </br> |
4. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue. </br> | 4. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue. </br> | ||
| − | 5. Prepare syringes with their cap and the reservoir (50 ml Falcon) </br> | + | 5. Prepare syringes with their cap and the reservoir (50 ml Falcon). </br> |
6. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white. </br> | 6. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white. </br> | ||
| − | 7. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer) </br> | + | 7. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer). </br> |
| − | 8. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer</br> | + | 8. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer.</br> |
| − | 9. Prepare into 10 Eppendorf tubes of 2 ml to aliquot pET43.1a(+) and pSB1C3.</br> | + | 9. Prepare into 10 Eppendorf tubes of 2 ml to aliquot pET43.1a(+) and pSB1C3.</br><br /> |
| − | Because we have only bench microfuges, we need to dispense our volume in smaller fractions. </br> | + | <font color = "red">  ⚠ Because we have only bench microfuges, we need to dispense our volume in smaller fractions. </font></br><br /> |
| − | 10. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes</br> | + | 10. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes.</br> |
| − | 11. Centrifuge (30 | + | 11. Centrifuge (30 minutes, 15 000g, at room temperature). </br> |
| − | 12. Add 3.5 ml of isopropanol, mix to precipitate the DNA</br> | + | 12. Add 3.5 ml of isopropanol, mix to precipitate the DNA.</br> |
| − | 13. Centrifuge (30 | + | 13. Centrifuge (30 minutes, 15 000g, at room temperature). </br> |
| − | 14. Remove isopropanol with 1 ml pipet without taking DNA and place into chemical waste container</br> | + | 14. Remove isopropanol with 1 ml pipet without taking DNA and place into chemical waste container.</br> |
| − | 15. Add 1 ml of 70% ethanol, centrifuge again (15 | + | 15. Add 1 ml of 70% ethanol, centrifuge again (15 minutes, 15 000g, at room temperature) and let air dry.</br> |
| − | 16. Resuspend in 50 ml of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C</br> | + | 16. Resuspend in 50 ml of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C.</br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
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<div class="lightbox" id="exp4"> | <div class="lightbox" id="exp4"> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> To linearize the different plasmids with appropriate enzymes. </br>We perform restriction enzyme digestion in order to recover linear backbones of the plasmids. We choose appropriate restriction sites based on the host plasmids.</br></br> | + | <h6><U> Aim:</U></h6> To linearize the different plasmids with appropriate enzymes. </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> | + | We perform restriction enzyme digestion in order to recover linear backbones of the plasmids. We choose appropriate restriction sites based on the host plasmids.</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/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a></br></br> |
| − | <U>Materials:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| + | <H6><U>Materials:</U></H6> | ||
• Restriction enzymes: Xba I, Hind III, Spe I, BamH I (New England Biolabs, NEB)</br> | • Restriction enzymes: Xba I, Hind III, Spe I, BamH I (New England Biolabs, NEB)</br> | ||
• Restriction enzyme buffers <br> | • Restriction enzyme buffers <br> | ||
• 37°C water bath</br> | • 37°C water bath</br> | ||
• UV spectrophotometer</br></br> | • UV spectrophotometer</br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | <U>Results:</U></br> | + | Measure the quantity of plasmid using a spectrophotometer (ultrospec 3100 pro, Pharmacia GE health care)</br></br> |
| + | <h6><U>Results:</U></h6></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 1 :</U> Absorbances </p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | </br></br><br /> | |
| − | </br></br>< | + | |
The concentrations measured after dilution were too low. We eventually switched to a Nanodrop (Thermofisher) because the plastic uvette gave too much background. </br> | The concentrations measured after dilution were too low. We eventually switched to a Nanodrop (Thermofisher) because the plastic uvette gave too much background. </br> | ||
</br> | </br> | ||
| − | <U>Method for digestion by Restriction enzymes</U></br> | + | <h6><U>Method for digestion by Restriction enzymes</U></h6></br> |
| − | 1. Mix all the reagents and let digest during 2 | + | 1. Mix all the reagents and let digest during 2 hours at 37°C. </br></br> |
| − | + | ||
| − | • pET43.1 a at 87.7 ng/µ will be digested by BamH I and Hind III (NEB)</br> | + | <font color = "red">   ⚠ Big volumes must be added first! </font></br><br /> |
| − | • pSB1C3 at 26.7 ng/µ will be digested by Spe I and Xba I (NEB)</br></br> | + | |
| + | • pET43.1 a at 87.7 ng/µl will be digested by BamH I and Hind III (NEB).</br> | ||
| + | • pSB1C3 at 26.7 ng/µl will be digested by Spe I and Xba I (NEB).</br></br> | ||
2. We began the digestion 17h20. Here we digest 400 ng of DNA. We doubled Hind III volumes because this enzyme has only 50% of efficiency in Custmart (NEB) buffer.</br></br> | 2. We began the digestion 17h20. Here we digest 400 ng of DNA. We doubled Hind III volumes because this enzyme has only 50% of efficiency in Custmart (NEB) buffer.</br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 2</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | 3. Store at -20°C</br> | + | 3. Store at -20°C. </br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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</div> | </div> | ||
</body> | </body> | ||
<html> | <html> | ||
