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<h2><B>Microbiology Notebook</B></h2>

<a href="#exp4"><h4> 8. Make a culture of DH5&alpha; pET43.1 to monitor the growth curve </h4></a></br>  

<a href="#exp6"><h4> Stab culture</h4></a></br>

<a href="#exp7"><h4> Resuspension of C1 and C2 and all inserts synthesized by iDT</h4></a></br>  

<a href="#exp8"><h4> 10. Extraction of pET43.1a(+) and pSB1C3 DNA from agarose gel </h4></a></br>

<a href="#exp9"><h4> 11. Digestion of inserts (C1 and C2)</h4></a></br>  

<a href="#exp10"><h4> 12. Dephosphorylation </h4></a></br>   

<a href="#exp11"><h4> 13. Ligation of C1 and C2 with pET43.1a(+) </h4></a></br>  

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             <U> Aim:</U> Check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.</br> Seed pET43.1 DH5&alpha; 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> 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 &micro;g/ml (LB + CB50) or with chloramphenicol 34 &micro;g/ml (LB + CM34) were counted for the presence of individual distinct colonies. </br></br>LB + CB50</br></br>

       <td><strong><p>pET43.1a(+) DH5&alpha;</p></strong></td>

       <td>0</td>

       <td>2</td>

       <td>23</td>

       <td><strong> <p>pET43.1a(+) DH5&alpha;</br> (different batch)</p></strong></td>

       <td>Uncountable (Overgrown)</td>

       <td>2</td>

       <td><strong><p>pET43.1a(+) DH5&alpha;</br> (different batch)</p></strong></td>

       <td>Uncountable </br>(Overgrown)</td>

       <td>15</td>

</br>

<center>Table 3</center></br> LB + CM34</br></br>

       <td><strong><p>pSB1C3 DH5&alpha;</p></strong></td>

       <td>1</td>

       <td>78</td>

       <td><strong> <p>pSB1C3 DH5&alpha;</br> (different batch)</p></strong></td>

       <td>38 </td>

       <td> Uncountable (Overgrown) </td>

</table>

</br>

<center>Table 4</center></br>  

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<U>What we did in the lab:</U></br>

           <U>Materials:</U>

&bull; Agarose <br>

&bull; Microwave oven /<br>

&bull; Precision balance </br>

 

</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>

2. Agarose gel: We prepared 0.7% w/v agarose gel.</br></br>

&bull; pSB1C3 plasmid (digested by SpeI/XbaI)<br>

&bull; pET43.1a plasmid digested by BamHI/HindIII</br>

       <td><strong><p>Sample </p></strong></td>

       <td>Molecular </br> weight</td>

       <td>pET43.1a(+)</br> uncut</td>

       <td></td>

       <td>pET43.1a(+)</br> B-H</td>

       <td></td>

       <td>pBS1C3</br> S-X</td>

       <td></td>

       <td>pBS1C3</br> uncut</td>

       <td><strong> </strong></td>

       <td>DNA (&micro;l)</td>

       <td> 5</td>

       <td> 2</td>

       <td></td>

       <td> 2</td>

       <td></td>

       <td> 4</td>

       <td></td>

       <td> 4</td>

       <td><strong> </strong></td>

       <td>H20</td>

       <td> 0</td>

       <td> 8</td>

       <td></td>

       <td>8</td>

       <td></td>

       <td>6</td>

       <td></td>

       <td>6</td>

       <td><strong> </strong></td>

       <td>6X</br>Loading</br>buffer</td>

       <td> 0</td>

       <td> 2</td>

       <td></td>

       <td>2</td>

       <td></td>

       <td>2</td>

       <td></td>

       <td>2</td>

</table>

</br>

<center>Table 5</center></br></br>   

<U>Results</U></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>

 

 

 

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               <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 link</br></br>

               <U>What we did in the lab:</U></br>

               <U>Materials:</U>

&bull; L.B (Luria Broth) medium autoclaved (5 ml) </br>

&bull; Antibiotic: Carbenicillin 100 mg/ml<br>

&bull; Cones for P10<br>

               <U>Method:</U></br>

1. Pick one colony and put it into 5 ml of LB medium + 2,5 of carbenicillin, to obtained 50

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               <U> Aim:</U> Use the preculture of June 13, 2016 to start the new culture.</br></br>

              <U> Protocol:</U> follow in this link</br></br>

               <U>What we did in the lab:</U></br>

               <U>Materials:</U></br>

&bull; LB autoclaved 500 ml<br>

&bull; swing bucket centrifuge (JOUAN GR4i)</br>

 

</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>

2. Add 5ml of fresh L.B, resuspend the pellet and centrifuge again at 3500 RPM during 7 min. </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>

5. Grow in the shaking incubator (T= 37 °C / 130 RPM) Overnight</br>

We overshot the 0.7 OD, setpoint therefore we continued overnight. </br>

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               <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 (OD600nm).</br></br>

              <U> Protocol:</U> follow in this link</br></br>

               <U>What we did in the lab:</U></br>

               <U>Materials:</U></br>

&bull; Falcon of 50ml </br>

&bull; L.B (Luria Broth) autoclaved (500 ml) </br>

&bull; Microbiology equipment</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>

1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at OD600nm every hour for the first three hours, then every 20 min onwards. We recorded OD600nm for this specific experiment after 3 hours.</br>

</br></br>

 

       <td><strong><p>12h37 </p></strong></td>

       <td></td>

       <td>0.013</td>

       <td>0.015</td>

       <td><strong> <p>13h07</p></strong></td>

       <td>0.023</td>

       <td> 0.027</td>

       <td><strong> <p>13h37</p></strong></td>

       <td>0.063</td>

       <td> 0.059 </td>

       <td><strong> <p>14h07</p></strong></td>

       <td>0.110 </td>

       <td> 0.105</td>

       <td><strong> <p>14h27</p></strong></td>

       <td>0.172 </td>

       <td> 0.170</td>

       <td><strong> <p>14h47</p></strong></td>

       <td>0.265 </td>

       <td> 0.256</td>

       <td><strong> <p>15h07</p></strong></td>

       <td>0.384 </td>

       <td> 0.377 </td>

       <td><strong> <p>15h27</p></strong></td>

       <td>0.491 </td>

       <td> 0.482</td>

       <td><strong> <p>15h47</p></strong></td>

       <td>0.567 </td>

       <td> 0.557</td>

       <td><strong> <p>16h06</p></strong></td>

       <td>0.697</td>

       <td> 0.608</td>

       <td><strong> <p>16h29</p></strong></td>

       <td>0.816</td>

       <td> 0.755</td>

       <td><strong> <p>17h03</p></strong></td>

       <td>0.954 </td>

       <td> 0.909 </td>

       <td><strong> <p>17h20</p></strong></td>

       <td>0.954</td>

       <td> 0.909</td>

       <td><strong> <p>17h40</p></strong></td>

       <td>1.061</td>

       <td> 1.029</td>

       <td><strong> <p>18h00</p></strong></td>

       <td>1.092</td>

       <td> 1.082</td>

       <td><strong> <p>18h00</p></strong></td>

       <td>1.122</td>

       <td> 1.132</td>

</br>

<center>Table 6</center></br> </br>

<U>Growth curve :</U>

 

<center></br> Figure 2: Growth curve for DH5&alpha;-pET43.1a(+) by measurement of  OD600nm. O-time point corresponds to 12h37</br></br></br></br></center>

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<U> Protocol: follow on this link</U></br></br>

<U> What we did on the lab:</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>

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>

4. Grow overnight at 37 °C and next day place it at 4°C</br>

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             <U> Aim:</U> we received our gene block synthetized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer. </br> Add TE buffer in each tubes and refer to the next table for volumes

       <th>Cfinal (ng/&micro ;l)</th>

       <th>V(TE) (&micro ;l)</th>

       <td><strong><p>A1 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

     <td><strong><p>A2 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

     <td><strong><p>C1 </p></strong></td>

       <td>1000</td>

       <td>10</td>

       <td>100</td>

     <td><strong><p>C2 </p></strong></td>

       <td>1000</td>

       <td>10</td>

       <td>100</td>

   <td><strong><p>D1 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>D2 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>E1 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>E2 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>B2 </p></strong></td>

       <td>1000</td>

       <td>10</td>

       <td>100</td>

</table>

</br>

<center>Table 7</center></br> </br>

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             <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. SpeI/ XbaI or BamHI/HindIII 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> Protocol:</U> follow in this link</br></br>

             <U>What we did in the lab:</U></br>

             <U>Material:</U></br>

&bull; We use the NEB extraction kit. Monarch DNA Gel Extraction Kit (/#T1020G) </br></br>

<U>Method:</U></br>

2. Add 4 volumes of gel dissolving buffer (NT1) to the gel slice. For NT1 volumes, refer to the Table 8: . </br>

       <td><strong><p>pET43.1a(+)</p></strong></td>

       <td>m1</td>

       <td>0.9</td>

       <td>1.3</td>

       <td>329.6</td>

       <td><strong><p>pET43.1a(+)</p></strong></td>

       <td>m2</td>

       <td>0.9</td>

       <td>1.3</td>

       <td>265.3</td>

     <td><strong><p>pSB1C3 </p></strong></td>

       <td>m1</td>

       <td>0.9</td>

       <td>1.2</td>

       <td>251.1</td>

     <td><strong><p> pSB1C3 </p></strong></td>

       <td>m2</td>

       <td>1.0</td>

       <td>1.2</td>

       <td>185.6</td>

<center>Table 8</center></br> </br>

3. Incubate the samples between 37-55°C vortexing periodically until the gel slice is completely dissolved (/ ~ 10min) </br>

4. Load sample onto the column. Close the cap, spin for 1 min then discard flow though</br>

5. Re insert column into collection tube. Add 200 µl DNA wash buffer and spin for one minute. Discard flow-through</br>

6. Repeat step 5</br>

7. Transfer column to a clean 1.5 ml microfuge tube</br>

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>

 

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             <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 link</br></br>

<U>Method:</U></br>

Add all reagents in 1 ml Eppendorf </br></br>

- Digest during 2 h at 37 °C </br>

- For the reagent volumes, refer to the Table 9. Total volume = 50 µl</br></br>

       <td><strong><p>DNA (&micro;l)</p></strong></td>

       <td>20</td>

       <td>20</td>

       <td><strong><p>BamHI (&micro;l)</p></strong></td>

       <td>1</td>

       <td>1</td>

    </tr>

     <td><strong><p>HindIII (&micro;l)</ </p></strong></td>

             <td>2</td>

       <td>2</td

     <td><strong><p> H20 (&micro;l)</ </p></strong></td>

       <td>22</td>

       <td>22</td>

</tr>

     <td><strong><p> CutSmart(&micro;l)</ </p></strong></td>

       <td>5</td>

       <td>5</td>

</br>

<center>Table 9</center></br> </br>

 

             <U> Aim:</U></br></br>

 

            <U> Protocol:</U> follow in this link</br></br>

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             <U> Aim:</U></br></br>

 

            <U> Protocol:</U> follow in this link</br></br>