Difference between revisions of "Team:Goettingen/Results"

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<img src=" https://static.igem.org/mediawiki/2015/5/58/PCR_RFP_DsRed_iGEM_Goettingen_2015.jpg" style="; float:right;">
 
<img src=" https://static.igem.org/mediawiki/2015/5/58/PCR_RFP_DsRed_iGEM_Goettingen_2015.jpg" style="; float:right;">
 
<p>After purification of the PCR product it was ligated into pJET1.2 by subcloning (blunt end ligation). This vector serves to clone the fluorescent protein without triggering its activity, which may interact with the expression vector or the chosen <em>E.coli </em>strain.</p>
 
<p>After purification of the PCR product it was ligated into pJET1.2 by subcloning (blunt end ligation). This vector serves to clone the fluorescent protein without triggering its activity, which may interact with the expression vector or the chosen <em>E.coli </em>strain.</p>
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<p>Ligation into pJET1.2 was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with <em>Kpn</em>I and <em>Sac</em>I (Fig. 2).</p>
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<p>Ligation into pJET1.2 was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and restricted with <em>Kpn</em>I and <em>Sac</em>I (Fig.2).</p>
 
<p>Once restriction controls showed the correct bands, both pJET_RFP_3 and pJET_RFP_7 were sent for sequencing by the G2L laboratory.</p>
 
<p>Once restriction controls showed the correct bands, both pJET_RFP_3 and pJET_RFP_7 were sent for sequencing by the G2L laboratory.</p>
 
<img src=" https://static.igem.org/mediawiki/2015/1/1d/Restr_pJET_RFP_DsRed_iGEM_Goettingen_2015.jpg" style="; float:right;">
 
<img src=" https://static.igem.org/mediawiki/2015/1/1d/Restr_pJET_RFP_DsRed_iGEM_Goettingen_2015.jpg" style="; float:right;">
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<a href="" onClick=" $('#menu3').slideToggle(300, function callback() {  }); return false;"><h1>Esterase and Phosphatase construct</h1></a>
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<p>ESTERASE AND PHOSPHATASE CONSTRUCTS</p>
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<p>Esterase was amplified from pET101_E064 and phosphatase from pTOPOXL_PLP07 by PCR for each enzyme (Fig.1). Both plasmids were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites <em>Kpn</em>I and <em>Sac</em>I for esterase and XhoI and PstI for the phosphatase in order to make them compatible for insertion into the multiple cloning site of the pBAD vector .The genes for these two enzymes had been found in screenings of metagenomic libraries.</p>
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<p>After purification of the PCR products, they were ligated into pJET by blunt end ligation. This vector serves to clone the enzymes without triggering their activity, which may interact with the vector or the <em>E.coli</em>. In the case of phosphatase, the PCR products needed to be purified by gel extraction, to eliminate left over template plasmids, which could interfere with the transformation.</p>
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<p>Ligation into pJET was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with a Quiaprep spin Miniprep kit and restricted with either <em>Kpn</em>I and <em>Sac</em>I or <em>Xho</em>I and <em>Pst</em>I, depending on the Enzyme (Fig.2&amp;3).</p>
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<p>Once restrictions showed the correct bands, both pJET containing esterase and phosphatase were sent for sequencing by the G2L laboratory.</p>
  
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Revision as of 08:08, 16 September 2015



Project Results

Transformation Efficiency Kit, RFP construct (iGEM)

RFP construct

Esterase and Phosphatase construct

Here you can describe the results of your project and your future plans.

What should this page contain?
  • Clearly and objectively describe the results of your work.
  • Future plans for the project
  • Considerations for replicating the experiments

Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

  • A list of linked bullet points of the successful results during your project
  • A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

Inspiration

See how other teams presented their results.