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| − | <div id="week1">
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| − | <p>June 6, 2016:…</p>
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| − | <p>
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| − | <a href="#exp1">Transformation of pSB1C3 and pET43.1a(+)</a></br>
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| − |
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| − | <p>June 7, 2016: …</p>
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| − | <p>
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| − | <a href="#exp2">Harvest the culture with Midiprep</a></br>
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| − | </p>
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| − |
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| − | <p>June 8, 2016: …</p>
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| − | <p>
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| − | <a href="#exp3">Extraction of plasmid DNA</a></br>
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| − | </p>
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| − |
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| − | <p>June 9, 2016: …</p>
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| − | <p>
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| − | <a href="#exp4">Digestion of the plasmid pSB1C3 and pET43.1a(+)</a></br>
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| − |
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| − |
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| − | <div class="lightbox" id="exp1">
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| − | <figure>
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| − | <a href="#" class="closemsg"></a>
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| − | <figcaption> <p><U> Aim:</U> To increase the amount of plasmid by transformation in competent cells. </br>
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| − | The amount of plasmid supplied is insufficient to perform all our future experiments. Therefore, we need to amplify the amount of plasmids. </br></br>
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| − |
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| − | <U> Protocol:</U> follow in this link
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| − | </br></br>
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| − | <U>What we did in the lab:</U>
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| − | </br>
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| − | <U>Materials:</U>
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| − | </br>
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| − | • subcloning competent cells</br>
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| − | • PSB1C3 plasmid (from shipped BioBrick-competent cells testing kit), chloramphenicol resistance</br>
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| − | • pET43.1a (GE health care), ampicillin resistance (or carbenicillin) </br>
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| − | • SOC (Super optimal Broth) media</br>
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| − | • LB (lysogeny Luria broth) Agar plates containing 50 g/ml carbenicillin or 34 g/ml chloramphenicol</br>
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| − | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etcfollow this link)
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| − | </br></br>
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| − | <U>Method:</U></br>
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| − | 1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells for 50 µl/vial.</br>
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| − | 2. Add 50 pg plasmids to each 50 µL of competent cells vial and tap gently.</br>
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| − | 3. Place on ice for 30 min. Meanwhile, warm LB agar plates from cold room in 37°C non shaking incubator.</br>
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| − | 4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 min.</br>
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| − | 5. Add 500 µL of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).</br>
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| − | 6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.</br>
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| − | 7. Near Bunsen burner flame, add 45 µL of competent cells +135µL of SOC or 200 µL aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.</br>
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| − | 8. Place plates inverted in the static 37°C incubator overnight. </br>
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| − | </p>
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| − | </figcaption>
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| − | </figure>
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| − | </div>
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| − |
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| − | <div class="lightbox" id="exp2">
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| − | <figure>
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| − | <a href="#" class="closemsg"></a>
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| − | <figcaption><p>
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| − | <U> Aim:</U> To start a culture for Miniprep. In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.
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| − | </br></br>
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| − |
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| − |
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| − | <U> Protocol:</U> follow in this link
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| − | </br></br>
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| − |
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| − | <U>What we did in the lab:</U>
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| − | </br>
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| − | <U>Materials:</U>
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| − | • Microbiology equipement </br>
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| − | • 25 ml flasks<br>
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| − | • Carbenicillin 50 mg/ml</br>
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| − | • Chloramphenicol 34 mg/ml</br>
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| − | • LB medium</br>
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| − | </br>
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| − | <U>Method:</U></br>
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| − | 1. One colony is picked from the plates and shaken in 25 ml of LB supplemented with Carbenicillin or Chloramphenicol at 50 g/ml or 34 g/ml respectively. </br>
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| − | 2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight. </br>
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| − | </p>
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| − | </figcaption>
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| − | </figure>
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| − | </div>
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| − |
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| − | <div class="lightbox" id="exp3">
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| − | <figure>
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| − | <a href="#" class="closemsg"></a>
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| − | <figcaption><p>
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| − | <U> Aim:</U> To perform a midiprep to isolate plasmid DNA of pSB1C3 and pET43.1a. </br>
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| − | The amplification method to increase the amount of plasmid is called Mini or Midiprep.
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| − |
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| − | </br></br>
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| − |
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| − |
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| − | <U> Protocol:</U> follow in this link
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| − | </br></br>
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| − |
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| − | <U>What we did in the lab:</U>
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| − | </br>
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| − | <U>Materials:</U>
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| − | • 50 ml Falcon tube</br>
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| − | • Shaking incubator (INFORS HT)<br>
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| − | • Carbenicillin 50 mg/ml</br>
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| − | • Swing bucket centrifuge (JOUAN GR41)</br>
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| − | • QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)</br>
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| − | </br>
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| − |
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| − | <U>Method:</U></br>
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| − | 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>
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| − |
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| − | 1. Use culture from overnight (17 hr) step on June 7, 2016 </br>
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| − | 2. Pour culture in 50 ml Falcon nd centrifuge (15 min, 3500g, 4°C) </br>
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| − | 3. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet </br>
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| − | 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>
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| − | 5. Prepare syringes with their cap and the reservoir (on 50 ml Falcon) </br>
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| − | 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>
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| − | 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>
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| − | 8. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer</br>
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| − | 9. Prepare 10 tubes of 2 ml to aliquot pET43.1 and pSB1C3. </br>
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| − | Because we have only bench microfuges, we need to dispense our volume in smaller fractions. </br>
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| − | 10. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes</br>
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| − | 11. Centrifuge (30 min, 15000g, room temperature) </br>
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| − | 12. Add 3.5 ml of isopropanol, mix to precipitate the DNA</br>
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| − | 13. Centrifuge (30 min, 15 000g, at RT) </br>
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| − | 14. Remove isopropanol with pipet without taking DNA and place into chemical waste container</br>
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| − | 15. Add 1 ml of 70% ethanol, centrifuge again (15 min, 15 000g, RT) and let air dry</br>
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| − | 16. Resuspend in 50 L of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C</br>
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| − | </p>
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| − | </figcaption>
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| − | </figure>
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| − | </div>
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| − |
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| − | <div class="lightbox" id="exp4">
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| − | <figure>
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| − | <a href="#" class="closemsg"></a>
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| − | <figcaption><p>
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| − | <U> Aim:</U> To linearize the different plasmids with appropriate enzymes. </br>
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| − | We perform restriction enzyme digestion in order to recover linear backbones of the plasmids. We choose appropriate restriction sites based on the host plasmids.
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| − |
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| − | </br></br>
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| − |
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| − |
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| − | <U> Protocol:</U> follow in this link
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| − | </br></br>
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| − |
| |
| − | <U>What we did in the lab:</U>
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| − | </br>
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| − | <U>Materials:</U></br>
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| − | • Restriction enzymes: XbaI, HindIII, SpeI, BamHI (New England Biolabs, NEB)</br>
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| − | • Restriction enzyme buffers <br>
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| − | • 37°C water bath</br>
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| − | • UV spectrophotometer</br>
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| − | </br>
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| − | <U>Method:</U></br>
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| − | Measure the quantity of plasmid using a spectrophotometer (utrospec 3100 pro, Pharmacia GE health care)</br>
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| − |
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| − | Results:
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| − |
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| − | <table>
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| − | <thead>
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| − | <tr>
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| − | <th>λ= 260 nm</th>
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| − | <th>pET43.1 a(+)</th>
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| − | <th>pSB1C3</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td><strong><p>A<sub>260</sub></p></strong></td>
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| − | <td>0.008</td>
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| − | <td>0.026</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>A<sub>280</sub></strong></td>
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| − | <td>0.001</td>
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| − | <td>0.014</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>A<sub>260</sub>/A<sub>280</sub></strong></td>
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| − | <td>4</td>
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| − | <td>1.85</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>C diluted</strong></td>
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| − | <td>0.4 ng/µl</td>
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| − | <td>1.9 ng/µl</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>C final</strong></td>
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| − | <td>26.7 ng/µl
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| − | </td>
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| − | <td>86.7 ng/µl
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| − | </em></td>
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| − | </tr>
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| − | </tbody>
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| − | </table>
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| − | Table 1
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| − | </br></br></br>
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| − |
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| − | The measured concentration after dilution were too low. We eventually switched to a Nanodrop (Thermofisher) because the plastic uvette gave too much background. </br>
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| − | </br>
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| − |
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| − | <U>Method for digestion by Restriction enzymes</U></br>
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| − | 1. Mix all the reagents and let digest during 2 hr at 37°C </br>
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| − | Big volumes must be added first!</br>
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| − |
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| − | • pET43.1 a at 87.7 ng/ will be digested by BamHI and Hind III (NEB)</br>
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| − | • pSB1C3 at 26.7 ng/ will be digested by SpeI and XbaI (NEB)</br>
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| − |
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| − | 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>
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| − | <table>
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| − | <thead>
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| − | <tr>
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| − | <th></th>
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| − | <th>pSB1C3 (26.7 ng/µl)</th>
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| − | <th>pET43.1 a (86.7 ng/µl)</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td><strong><p>Vol SpeI </p></strong></td>
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| − | <td>1 µl</td>
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| − | <td>0 µl</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>Vol XbaI </strong></td>
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| − | <td>1 µl</td>
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| − | <td>0 µl</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>Vol HindIII </strong></td>
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| − | <td>0 µl </td>
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| − | <td>2 µl</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>Vol BamHI </strong></td>
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| − | <td>0 µl</td>
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| − | <td>1 µl</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong>Vol H2O</strong></td>
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| − | <td>28 µl</td>
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| − | <td>37.4 µl</em></td>
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| − | <tr>
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| − | <td><strong>Vol buffer 10X (Cutsmart) </strong></td>
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| − | <td>5 µl</td>
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| − | <td>5 µl</td>
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| − | </tr>
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| − | <td><strong>Vol TOTAL </strong></td>
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| − | <td>50 µl</td>
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| − | <td>50 µl</td>
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| − | </tr>
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| − | </tr>
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| − | </tbody>
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| − | </table>Table 2
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| − |
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| − | 3. Store at -20°C</br>
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| − |
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| − | </p>
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| − | </figcaption>
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| − | </figure>
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| − | </div>
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