Team:Goettingen/week10-2

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                                <!-- Text body -->
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Language: <img height="20", src="http://www.patrickreinke.de/igem/eng.jpg"> English, <img height="20", src="http://www.patrickreinke.de/igem/deu.jpg"> <a href="https://2012.igem.org/Team:Goettingen/main_deu">Deutsch</a> <br>
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Line 532: Line 21:
<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
-
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>
+
In order to clone the <i>flhDC</i>-promoter constructs into pSB1C3, all components were digested with <i>EcoRI</i> and <i>PstI</i> according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. </li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
-
The gel did not feature fragments of the expected size.Obviously, something was wrong with the digestion. </li>
+
The gel did not feature fragments of the expected size. Obviously, something was wrong with the digestion. </li>
         </ul>
         </ul>
<br>
<br>
Line 541: Line 30:
                 <li>Experiment:  <br>
                 <li>Experiment:  <br>
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>
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>
 +
<br>
20E - #2 <br>
20E - #2 <br>
20G - #1 <br>
20G - #1 <br>
Line 549: Line 39:
18K - #1 <br>
18K - #1 <br>
18C - #2 <br></li>
18C - #2 <br></li>
 +
<br>
     </ul>
     </ul>
<br></td></tr>
<br></td></tr>
Line 569: Line 60:
<ul>
<ul>
<li>Experiment:  <br>
<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 Pfu-Polymerase according to the following protocol. The success of the PCR was subsequently investigated preparing a 1% agarose gel.<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 <i>pfu</i> polymerase according to the following protocol. The success of the PCR was subsequently investigated preparing a 1% agarose gel.<br>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
The PCR failed for no bands were visible on the gel whereas the marker was clearly discernible.</li>
The PCR failed for no bands were visible on the gel whereas the marker was clearly discernible.</li>
Line 586: Line 77:
<ul>
<ul>
<li>Experiment: <br>
<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 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>
+
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 <i>pfu</i> polymerase according to the following <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>l. 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>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
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>
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>
Line 602: Line 93:
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
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>
+
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 <i>pfu</i> polymerase according to the following <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> one last time. Here again, a 1% agarose gel was prepared to investigate the PCRs outcome.</li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
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>
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>
Line 610: Line 101:
<ul>
<ul>
<li>Experiment:  <br>
<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>
+
The digestion of the <i>flhDC</i>-promoter constructs <i>EcoRI</i> and <i>PstI</i> 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>
<li>Observations & Results: <br>
The digestion was successful. Bands of the expected size could be observed and cut out.</li>
The digestion was successful. Bands of the expected size could be observed and cut out.</li>
Line 627: Line 118:
<ul>
<ul>
<li>Experiment: <br>
<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 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>
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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 Phusion polymerase according to the following <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. 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>
<li>Observations & Results: <br>
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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>
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Finally,the PCR was successful! We were able to receive bands of the expected size for each construct. Obviously, the <i>pfu</i> polymerase was not functional anymore and had caused the PCRs failure. </li>
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<b>Important pages</b>:<br>
 
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<a href="https://2012.igem.org/Team:Goettingen">Home</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Modeling">Modeling</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Notebook">Notebook</a>;
 
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Latest revision as of 17:18, 22 September 2012

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#2 Speed Improvement - 10th week

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V07_02


V07_02_1 Preparative double digestion of 20E-flhDC, 2G-flhDC, 18O-flhDC and pSB1C3
  • Experiment:
    In order to clone the flhDC-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.
  • Observations & Results:
    The gel did not feature fragments of the expected size. Obviously, something was wrong with the digestion.

V07_02_2 Preparation of over night cultures
  • Experiment:
    In order to gain more plasmid material of the flhDC-promoter constructs over night cultured were prepared for a subsequent plasmid isolation.

    20E - #2
    20G - #1
    2G - #1
    20I - #2
    18M - #2
    18O - #2
    18K - #1
    18C - #2



V07_03


V07_03_1 Miniprep of the flhDC-promoter constructs
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.


V07_03_2 Amplification of the genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH
  • Experiment:
    The genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH were amplified by pfu polymerase according to the following protocol. The success of the PCR was subsequently investigated preparing a 1% agarose gel.
  • Observations & Results:
    The PCR failed for no bands were visible on the gel whereas the marker was clearly discernible.


V07_04


Repetition of the amplification of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH
  • Experiment:
    The genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH were amplified by pfu polymerase according to the following protocoll. 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.
  • Observations & Results:
    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.


V07_05


V07_05_1 Repetition of the amplification of the genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH
  • Experiment:
    To give it a last try the fliC (DH10B), fliC (Salmonella), motA, motB and yhjH 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.
  • Observations & Results:
    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.

V07_05_2 Repetition of the preparative double digestion of 20G-flhDC, 20I-flhDC, 18K-flhDC, and 18C-flhDC
  • Experiment:
    The digestion of the flhDC-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.
  • Observations & Results:
    The digestion was successful. Bands of the expected size could be observed and cut out.


V07_06


Repetition of the amplification of the fliC (DH10B), fliC (Salmonella), motA, motB and yhjH using the new polymerase
  • Experiment:
    The genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH 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.
  • Observations & Results:
    Finally,the PCR was successful! We were able to receive bands of the expected size for each construct. Obviously, the pfu polymerase was not functional anymore and had caused the PCRs failure.


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