Difference between revisions of "Team:Pasteur Paris/Microbiology week2"
| Line 330: | Line 330: | ||
<figcaption><p> | <figcaption><p> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
| − | <U>Materials:</U> | + | <U>Materials:</U></br> |
• 1X TAE buffer </br> | • 1X TAE buffer </br> | ||
• Agarose <br> | • Agarose <br> | ||
| Line 445: | Line 445: | ||
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></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></br> |
• 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> | ||
| Line 458: | Line 458: | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | 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> | ||
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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 LB, resuspend the pellet and centrifuge again at 3500 RPM during 7 min. </br> | 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 µl of carbenicillin 100 mg/ml</br> | + | 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) | + | 5. Grow in the shaking incubator (T= 37 °C / 130 RPM) overnight.</br> |
</br></br> | </br></br> | ||
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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 µl of carbenicillin 50 mg/ml added, swirl to mix</br> | + | 1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 µl of carbenicillin 50 mg/ml added, swirl to mix.</br> |
| − | 2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total) </br> | + | 2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total). </br> |
| − | 3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times</br> | + | 3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times.</br> |
| − | 4. Grow overnight at 37 °C and next day place it at 4°C</br> | + | 4. Grow overnight at 37 °C and next day place it at 4°C.</br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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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> Add TE (Tris 10 mM pH 8.0 EDTA 1 mM) buffer in each tubes and refer to the next table for volumes | + | <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> |
| + | <U>Method:</U></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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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> We want to have only the DNA backbone of pET43.1a(+) and pSB1C3, not the inserts that lie between the restriction sites. Spe I/ Xba I or BamH I/Hind III respectively, so we have to extract the plasmid backbone on out of an agarose gel. Furthermore, to clean the pET43.1a(+) and pSB1C3 backbones we use the gel extraction kit of QIAGEN. Afterwards we ligated our plasmid with the inserts. In this experiment we use inserts C1 and C2 | + | <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> | ||
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1. Excise the DNA fragment from the agarose gel and transfer to 1 ml Eppendorf tube. </br> | 1. Excise the DNA fragment from the agarose gel and transfer to 1 ml Eppendorf tube. </br> | ||
| − | 2. Add 4 volumes of gel dissolving buffer (NT1) to the gel slice. For NT1 volumes, refer to the Table 8: | + | 2. Add 4 volumes of gel dissolving buffer (NT1) to the gel slice. For NT1 volumes, refer to the Table 8: </br> |
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<U>Material:</U></br></br> | <U>Material:</U></br></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Add all reagents in 1 ml Eppendorf </br></br> | + | 1. Add all reagents in 1 ml Eppendorf </br></br> |
| − | + | 2. Digest during 2 h at 37 °C </br></br> | |
| − | + | 3. For the reagent volumes, refer to the Table 9. Total volume = 50 µl</br></br> | |
Revision as of 13:10, 17 October 2016
