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| | Here again a certain diversity in the growth rate could be observed. Whereas some cultures had already reached and optical density of 0.8, others were still at merely 0.04.Here again <i>fliC</i> featured a low density, this time in all strains and DH10B and BL21 show a general decreased growth rate. Striking is the fact that the strains that featured the best motility as well as the construct that seems to have the biggest influence show decreased division rate. Whether this phenomenon is a coincidence or not we are not sure about yet. | | Here again a certain diversity in the growth rate could be observed. Whereas some cultures had already reached and optical density of 0.8, others were still at merely 0.04.Here again <i>fliC</i> featured a low density, this time in all strains and DH10B and BL21 show a general decreased growth rate. Striking is the fact that the strains that featured the best motility as well as the construct that seems to have the biggest influence show decreased division rate. Whether this phenomenon is a coincidence or not we are not sure about yet. |
| | At the following day (26th of July) as well as the day after (27th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed. Almost a weak later (30th of July) still no swimming or chemotaxis had taken place, solely growth. However, the two colonies that had been applied into the agar not onto featured little swimming. Nevertheless, also these results are lousy considering the long time span. (</li> | | At the following day (26th of July) as well as the day after (27th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed. Almost a weak later (30th of July) still no swimming or chemotaxis had taken place, solely growth. However, the two colonies that had been applied into the agar not onto featured little swimming. Nevertheless, also these results are lousy considering the long time span. (</li> |
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| - | </table>
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| - | <table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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| - | <h2><b>V07_02 </b></h2><br>
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| - | <b>V07_02_1 Preparative double digestion of 20E-<i>flhDC</i>, 2G-<i>flhDC</i>, 18O-<i>flhDC</i> and pSB1C3</b><br>
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| - | <ul>
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| - | <li>Experiment: <br>
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| - | In order to clone the <i>flhDC</i>-promoter constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. </li>
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| - | <li>Observations & Results: <br>
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| - | The gel did not feature fragments of the expected size.Obviously, something was wrong with the digestion. </li>
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| - | </ul>
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| - | <br>
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| - | <b>V07_02_2 Preparation of over night cultures</b><br>
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| - | <ul>
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| - | <li>Experiment: <br>
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| - | In order to gain more plasmid material of the <i>flhDC</i>-promoter constructs over night cultured were prepared for a subsequent plasmid isolation.<br>
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| - | 20E - #2 <br>
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| - | 20G - #1 <br>
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| - | 2G - #1 <br>
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| - | 20I - #2 <br>
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| - | 18M - #2 <br>
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| - | 18O - #2 <br>
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| - | 18K - #1 <br>
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| - | 18C - #2 <br></li>
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| - | </ul>
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| - | <br></td></tr>
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| - | </table>
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| - |
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| - | <h2><b>V07_03 </b></h2><br>
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| - | <b>V07_03_1 Miniprep of the <i>flhDC</i>-promoter constructs</b><br>
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| - | <ul>
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| - | <li>Experiment: <br>
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| - | Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.</li></ul>
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| - | <br>
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| - | <br>
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| - | <b>V07_03_2 Amplification of the genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i></b><br>
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| - | <ul>
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| - | <li>Experiment: <br>
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| - | The genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Pfu-Polymerase according to the following protocol. The success of the PCR was subsequently investigated preparing a 1% agarose gel.<br>
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| - | <li>Observations & Results: <br>
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| - | The PCR failed for no bands were visible on the gel whereas the marker was clearly discernible.</li>
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| - | </ul><br>
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| - | </td></tr>
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| - | </table>
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| - | <table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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| - | <td width="900" bordercolor="black" valign="top">
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| - | <h2><b>V07_04 </b></h2><br>
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| - | <b> Repetition of the amplification of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> </i></i></b><br>
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| - | <ul>
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| - | <li>Experiment: <br>
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| - | The genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Pfu-Polymerase according to the following protocol. Additionally, a negative control as well as a positive control were prepared this time. The success of the PCR was subsequently investigated preparing a 1% agarose gel.<br></li>
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| - | <li>Observations & Results: <br>
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| - | The did not function again for no bands were visible on the gel; only the marker was clearly discernible. However, since also the positive control did not feature the slightest band, we assume that something might be wrong with our polymerase.</li>
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| - | <br></td></tr>
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| - | </table>
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| - | <br>
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| - |
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| - | <table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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| - | <tr bordercolor="black" valign="top">
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| - | <td width="900" bordercolor="black" valign="top">
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| - |
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| - | <h2><b>V07_05 </b></h2><br>
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| - | <b>V07_05_1 Repetition of the amplification of the genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i></b><br>
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| - | <ul>
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| - | <li>Experiment: <br>
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| - | To give it a last try the <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Pfu-Polymerase according to the following protocol one last time. Here again, a 1% agarose gel was prepared to investigate the PCRs outcome.</li>
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| - | <li>Observations & Results: <br>
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| - | The gel of the PCR delivered the same picture as last time. No band could be observed except for the marker. Even the positive control does not work. Thus, we decided to buy a new polymerase.</li>
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| - | </ul>
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| - | <br>
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| - | <b>V07_05_2 Repetition of the preparative double digestion of 20G-<i>flhDC</i>, 20I-<i>flhDC</i>, 18K-<i>flhDC</i>, and 18C-<i>flhDC</i></b><br>
| |
| - | <ul>
| |
| - | <li>Experiment: <br>
| |
| - | The test digestion of the <i>flhDC</i>-promoter constructs EcoRI and PstI were applied according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments.<br>
| |
| - | <li>Observations & Results: <br>
| |
| - | The digestion was successful. Bands of the expected size could be observed and cut out.</li>
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| - | </ul><br>
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| - | </td></tr>
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| - | </table>
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| - | <br>
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| - | <table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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| - | <tr bordercolor="black" valign="top">
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| - | <td width="900" bordercolor="black" valign="top">
| |
| - | <h2><b>V07_06 </b></h2><br>
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| - | <b> Repetition of the amplification of the <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> using the new polymerase </b><br>
| |
| - | <ul>
| |
| - | <li>Experiment: <br>
| |
| - | The genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Phusion-Polymerase according to the following protocol. We also prepared a negative control as well as a positive control. The success of the PCR was subsequently investigated preparing a 1% analytical agarose gel.<br></li>
| |
| - | <li>Observations & Results: <br>
| |
| - | Finally,the PCR was successful! We were able to receive bands of the expected size for each construct. Obviously, the old Pfu-polymerase was not functional anymore and had caused the PCRs failure. </li>
| |
| | <br></td></tr> | | <br></td></tr> |
| | </table> | | </table> |
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