Team:Warsaw/Calendar-Main/25 June 2008

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

Piotr, Weronika

Inoculation of 10ml LB with E.coli TOP10 carrying pZC.
Isolation and chemotransformation with pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
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

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
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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-<h3>Preparation of constructs with OmpA protein fusions</h3>
+<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>
+<h4>Piotr, Weronika</h4>
+<ol>
+<li>Inoculation of 10ml LB with E.coli TOP10 carrying pZC.</li>
+<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>
+<li>Plating of chemotransformants on LB+ampicillin.
+</li></ol>
+<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
 <h4>Michał K.</h4>
-<p>Repetition of PCRs and gel-out isolations from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/16_June_2008">16 June.</a>
+<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>
 </p>
-<h3>PCR of Alpha-link and link-A</h3>
+<h3>Preparation of alpha-A and omega-A fusions</h3>
 <h4>Michał L. Marcin and Ewa</h4>
 <ol>
-<li>We've ran 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. <br>Annealing temp 48&deg;C - 55&deg;C.<br>
+<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.
- Elongation time 1 - 2 minutes.</li>
+<ul><li>DNA template: pDRIVE-TAPTAG</li>
+<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>
+<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>
-<li>We have successfully amplified Alpha-link and link-A. Now its time for PCL (Polymerase Chain Ligation) to create fusions  Alpha-A and Omega-A (Fig.1.).</li></ol>
+<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/8/89/PCR_gradient_Alinker_WAW.jpg" width=400/></a>
+<var> <b>Fig. 1.</b> PCR product - link-A.</var>
-<img src="https://static.igem.org/mediawiki/2008/8/89/PCR_gradient_Alinker_WAW.jpg" width=300/>
-<var> <b>Fig. 1.</b> PCR product - gene coding A protein.</var>
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