Team:Warsaw/Calendar-Main/20 June 2008

From 2008.igem.org

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<h3>Cloning of protein Z DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b-OmpA-alpha</a> in place of OmpA</h3><h4>Piotr</h4>
 
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<p><ol><li>Result of ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-alpha </a> with protein Z DNA: 16 colonies grown.</li>
 
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<li>Each colony cultured overnight in LB + ampicilin.</li>
 
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<h4>Michał K.</h4>
<h4>Michał K.</h4>
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<li>Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with PstI (Orange buffer).</li>
<li>Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with PstI (Orange buffer).</li>
<li>Gel electrophoresis (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/20_June_2008#fig1">Fig. 1 and 2)</a>, no proper colonies found.</li></ol>
<li>Gel electrophoresis (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/20_June_2008#fig1">Fig. 1 and 2)</a>, no proper colonies found.</li></ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/78/Omp_control_digest_WAW.jpg" width=400/></a>
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<var><b>Fig. 1.</b> Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_alpha fusion with PstI.</var>
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<img src="https://static.igem.org/mediawiki/2008/1/1a/Omp_control_digest2_WAW.jpg" width=400/>
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<var><b>Fig. 2.</b> Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_omega fusion with PstI.</var>
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<h3>Preparation of alpha-A and omega-A fusions</h3>
<h3>Preparation of alpha-A and omega-A fusions</h3>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/78/Omp_control_digest_WAW.jpg" width=400/></a>
 
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<var><b>Fig. 1.</b> Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_alpha fusion with PstI.</var>
 
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<img src="https://static.igem.org/mediawiki/2008/1/1a/Omp_control_digest2_WAW.jpg" width=400/>
 
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<var><b>Fig. 2.</b> Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_omega fusion with PstI.</var>
 
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Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest of isolated plasmids with PstI (Orange buffer).
  3. Gel electrophoresis (Fig. 1 and 2), no proper colonies found.
Fig. 1. Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_alpha fusion with PstI. Fig. 2. Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_omega fusion with PstI.

Preparation of alpha-A and omega-A fusions

Michał L., Ewa, Marcin

PCR
ProductTemplatePrimersProduct length
linker-ApDRIVE-TapTagAL+link10+homo2 and AP+NotI 470 bp
alpha-linkerpUC19 AlphaL+SacI and AlphaP+link10+homo2600 bp
omega-linkerpUC19OmegaL+SacI and OmegaP+link10+homo2400 bp



Universal PCR program for protein A, alpha and omega
TemperatureTime
94°C4:00
94°C0:3028 cycles
50°C0:45
72°C2:00
72°C10:00
4°Cinfinite

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