Team:Goettingen/week11-2

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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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<h2><b>V07_02 </b></h2><br>
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<h2><b>V07_10 </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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<b>V07_10_1 Preparative double digestion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pUC18 </b><br>
<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
-
In order to clone the <i>flhDC</i>-promoter constructs intro 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 purification of the desired fragments. </li><br>
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In order to clone the new genes into pUC18, all components were digested with <i>EcoRI</i> and <i>XbaI</i> according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The restricted plasmid pUC18 was subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. The other digestion products were investigated via preparation of an 1% analytical gel and directly purified using the peqGOLD Gel Extraction Kit (Peqlab) according to the manual as well. </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 digestion was successful. For all PCR products as well as for the vector band of the expected size could be obtained and the fragments can now be used for further cloning. </li>
         </ul>
         </ul>
<br>
<br>
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<b>V07_02_2 Preparation of over night cultures</b><br>
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<b>V07_10_2 Insertion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> into pUC18</b><br>
         <ul>
         <ul>
                 <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>
+
The ligation of the digested genes with the plasmid pUC18 was conducted as described in the protocol.
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20E - #2 <br>
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</li>
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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>
     </ul>
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<h2><b>V07_11 </b></h2><br>
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<h2><b>V07_03 </b></h2><br>
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<b> Chemical transformation of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> into <i>E. coli</i> (DH10B)  </b><br>
-
<b>V07_03_1 Miniprep of the <i>flhDC</i>-promoter constructs</b><br>
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.</li></ul>
+
For the chemical transformation the standard protocol was followed. <br></li>
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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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<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>
+
<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 transformation was successful since all plates showed numeours colonies except the negative control. </li>
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<h2><b>V07_04 </b></h2><br>
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<h2><b>V07_12 </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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<b> Preparation of over night cultures</b><br>
<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>
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Over night cultures of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18 were prepared in order to isolate the plasmids. <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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<h2><b>V07_05 </b></h2><br>
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<h2><b>V07_13 </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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<b>V07_13_1 Miniprep of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18</b><br>
<ul>
<ul>
<li>Experiment: <br>
<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><br>
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Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.</li></ul>
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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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<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>
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<b>V07_13_2 Test digestion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18</b><br>
<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
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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>
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All vectors were digested with <i>EcoRI</i> and <i>XbaI</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 determine successfully ligated plasmids.<br>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
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The digestion was successful. Bands of the expected size could be observed and cut out.</li>
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Except for <i>fliC</i> (Salmonella) #1 all picked colonies seem to host plasmids containing the aimed insert. </li>
</ul><br>
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<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>
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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 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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<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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<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/Team">Team</a>;
 
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<a href="https://igem.org/Team.cgi?year=2012">Official Team Profile</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Project">Project</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Parts">Parts submitted to the Registry</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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<a href="https://2012.igem.org/Team:Goettingen/Saftey">Saftey</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Attributions">Attributions</a>
 
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Latest revision as of 17:18, 22 September 2012

Deutsch  / English 

#2 Speed Improvement - 11th week

Back to overview

V07_09


Purification of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH
  • Experiment:
    The amplicons were separated via application on an 1% agarose gel and subsequently cut out. The DNA was purified out of the gel slices using peqGOLD Gel Extraction Kit (Peqlab) according to the user manual.


V07_10


V07_10_1 Preparative double digestion of fliC (DH10B), fliC (Salmonella), motA, motB, yhjH and pUC18
  • Experiment:
    In order to clone the new genes into pUC18, all components were digested with EcoRI and XbaI according to the protocol. The restricted plasmid pUC18 was subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. The other digestion products were investigated via preparation of an 1% analytical gel and directly purified using the peqGOLD Gel Extraction Kit (Peqlab) according to the manual as well.
  • Observations & Results:
    The digestion was successful. For all PCR products as well as for the vector band of the expected size could be obtained and the fragments can now be used for further cloning.

V07_10_2 Insertion of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH into pUC18
  • Experiment:
    The ligation of the digested genes with the plasmid pUC18 was conducted as described in the protocol.


V07_11


Chemical transformation of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH into E. coli (DH10B)
  • Experiment:
    For the chemical transformation the standard protocol was followed.
  • Observations & Results:
    The transformation was successful since all plates showed numeours colonies except the negative control.


V07_12


Preparation of over night cultures
  • Experiment:
    Over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18 were prepared in order to isolate the plasmids.


V07_13


V07_13_1 Miniprep of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V07_13_2 Test digestion of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18
  • Experiment:
    All vectors were digested with EcoRI and XbaI according to the protocol. The restriction products were subsequently separated on a gel in order to determine successfully ligated plasmids.
  • Observations & Results:
    Except for fliC (Salmonella) #1 all picked colonies seem to host plasmids containing the aimed insert.


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