Team:Warsaw/Calendar-Main/25 June 2008

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<h3>Change of the reporter from <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> with B-galactosidase to GFP or RFP</h3>
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<h4>Piotr, Weronika</h4>
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<ol>
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<li>Inoculation of 10ml LB with E.coli TOP10 carrying pZC.</li>
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<li>Isolation and chemotransformation with <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A2>pSB1A2</a> standard plasmids carrying parts: <A href=http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840>BBa_E0840</a>(GFP genetrator) and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450>BBa_J04450</a> (RFP generator).</li>
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<li>Plating of chemotransformants on LB+ampicillin.
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</li></ol>
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
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<h4>Michał K.</h4>
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<p>Repetition of PCRs and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out purifications</a> from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/16_June_2008">16 June.</a>
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<p>'''Preparation of pMPMT5-AID+AIDT7 construct''' <br>
 
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1. Digest of isolated PCR product (from previous day) and pMPMT5-AID with HindIII and XbaI, dephosphorylation of pMPMT5-AID with CIAP <br>
 
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2. Clean-up of digested PCR product. <br>
 
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3. Gel electrophoresis of digested pMPMT5-AID and gel-out of proper band (4800 bp)<br>
 
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4. Overnight ligation of purified DNA fragments
 
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<h3>Preparation of alpha-A and omega-A fusions</h3>
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<h4>Michał L. Marcin and Ewa</h4>
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<ol>
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<li>We've run gradient <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain Alpha-link and link-A.
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<ul><li>DNA template: pDRIVE-TAPTAG</li>
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<li>Taq polymerase</li><li>buffer suplemented with (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub></li><li>MgCl<sub>2</sub> concentration gradient (from 1 to 4 mM)</li><li>annealing temp 55&deg;C</li><li>elongation time 1 - 2 minutes</li></ul></li>
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<li>We have successfully amplified Alpha-link and link-A (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_June_2008#fig1">Fig. 1</a>). Now it's time for PCL (Polymerase Chain Ligation) to create fusions  Alpha-A and Omega-A.</li></ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/8/89/PCR_gradient_Alinker_WAW.jpg" width=400/></a>
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<var> <b>Fig. 1.</b> PCR product - link-A.</var>
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Latest revision as of 14:02, 28 October 2008

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Change of the reporter from pZC320 with B-galactosidase to GFP or RFP

Piotr, Weronika

  1. Inoculation of 10ml LB with E.coli TOP10 carrying pZC.
  2. Isolation and chemotransformation with pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
  3. Plating of chemotransformants on LB+ampicillin.

Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

Repetition of PCRs and gel-out purifications from 16 June.

Preparation of alpha-A and omega-A fusions

Michał L. Marcin and Ewa

  1. We've run gradient PCR to obtain Alpha-link and link-A.
    • DNA template: pDRIVE-TAPTAG
    • Taq polymerase
    • buffer suplemented with (NH4)2SO4
    • MgCl2 concentration gradient (from 1 to 4 mM)
    • annealing temp 55°C
    • elongation time 1 - 2 minutes
  2. We have successfully amplified Alpha-link and link-A (Fig. 1). Now it's time for PCL (Polymerase Chain Ligation) to create fusions Alpha-A and Omega-A.
Fig. 1. PCR product - link-A.

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