Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602024"

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<h3>K1602024 - HistidinTag-Green fluorescent protein (His-GFP)</h3>
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<h3>K1602024 - HistidinTag-Green fluorescent protein-SilicaTag4 (His-GFP-Si4)</h3>
  
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<a href=” https://2015.igem.org/Team:TU_Darmstadt/Notebook/sec1/K1602021”title="Opens internal link in current window" class="internal link">K1602021</a> was used as template for SOE-PCR with oligonucleotides VF2 and 33 (Figure 1). The silica-tag Si4 was added using another SOE-PCR with oligonucleotides VF2 and VR (Figure 2). The PCR product was purified and digested with <em>EcoR</em>I and <em>Pst</em>I. The construct was ligated into pSB1C3 and transformed into <em>E. coli</em> Top 10 via heat shock. Colonies were screened by colony PCR with VF2 and VR. </p>
  
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Competent <em>E. coli </em>Top 10 cells were retransformed via heat shock with pSB1C3 harbouring <em> GFP</em> provided by the iGEM Registry. The plasmids were extracted and used as templates for a PCR with oligonucleotides 42 and VR. The PCR product was verified by gel electrophoresis (Figure 1) and digested with <em> Dpn</em>I. The purified product was digested with <em>EcoR</em>I and <em>Pst</em>I and ligated into pSB1C3. Competent <em>E. coli</em> Top 10 cells were transformed via heat shock and colonies were screened by colony PCR with oligonucleotides VF2 and VR (Figure 2).
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<figcaption><br><b>Figure 1</b> SOE PCR of His-GFP (1). The size of the amplified product 1 was around 0.9 kbp. DNA marker: 2-Log DNA Ladder.
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<figcaption><br><b>Figure 1</b> Standard PCR of Prefix-His-Tag-GFP (3). The size of the amplified product was around 0.9 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
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<figcaption><br><b>Figure 2</b> : Colony PCR of Prefix-His-Tag-GFP (1+2). Colonies 1 and 2 contained the insert of around 0.9 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
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<figcaption><br><b>Figure 2</b> SOE PCR of His-GFP-Si4 (1+2). The size of the amplified product 2 was around 1.1 kbp. DNA marker: 2-Log DNA Ladder.
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Latest revision as of 20:27, 17 September 2015

K1602024 - HistidinTag-Green fluorescent protein-SilicaTag4 (His-GFP-Si4)

K1602021 was used as template for SOE-PCR with oligonucleotides VF2 and 33 (Figure 1). The silica-tag Si4 was added using another SOE-PCR with oligonucleotides VF2 and VR (Figure 2). The PCR product was purified and digested with EcoRI and PstI. The construct was ligated into pSB1C3 and transformed into E. coli Top 10 via heat shock. Colonies were screened by colony PCR with VF2 and VR.


Figure 1 SOE PCR of His-GFP (1). The size of the amplified product 1 was around 0.9 kbp. DNA marker: 2-Log DNA Ladder.

Figure 2 SOE PCR of His-GFP-Si4 (1+2). The size of the amplified product 2 was around 1.1 kbp. DNA marker: 2-Log DNA Ladder.