Team:Warsaw/Calendar-Main/2 July 2008

From 2008.igem.org

(Difference between revisions)
 
(17 intermediate revisions not shown)
Line 1: Line 1:
{{WarNotebook}}
{{WarNotebook}}
<!-- do not edit above me! -->
<!-- do not edit above me! -->
 +
<html>
 +
<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, Emilia</h4>
 +
<ol>
-
<p>'''Preparation of constructs with OmpA protein fusions'''<br>
+
<li>
-
1. PCR on OmpA_linker and linker_alpha  with
+
Gel electrophoresis and gel-out of proper bands.
-
<html>
+
</li>
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP_XB">AlphaP_XB</a>
+
-
</html>
+
-
primers (20 cycles, annealing length 1.15 min, annealing temperature 57 degrees)<br>
+
-
2. PCR on OmpA_linker and linker_omega with
+
-
<html>
+
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP_EPB">OmegaP_EPB</a>
+
-
</html>
+
-
primers (20 cycles, annealing length 1.15 min, annealing temperature 57 degrees)<br>
+
-
3. Gel electrophoresis of PCR products and gel-out of OmpA_alpha and OmpA_omega <br>
+
-
4. Overnight digest of purified OmpA_alpha and OmpA_omega DNA with NdeI and BamHI <br>
+
-
5. Overnight digest of pET15b plasmid with NdeI and BamHI, dephosphorylation with CIAP
+
-
</p>
+
 +
<li>
 +
Ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> with standard 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>Chemotransformation of E.coli TOP10 with ligation products.</li>
 +
<li>Plating transformants on LB+Amp30+X-gal+IPTG.</li></ol></p>
 +
 +
 +
 +
<h3>Preparation of constructs on pET15b: OmpA_alpha and OmpA_omega</h3>
 +
<h4>Paweł</h4>
 +
<p><ol>
 +
 +
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of overnight digest.</li>
 +
<li> <A HREF="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</A> of OmpA_alpha (from Michał K.) and <a href=http://www.emdbiosciences.com/docs/docs/PROT/TB045.pdf>pET15b</a>. </li>
 +
<li> <A HREF="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</A> of OmpA_omega (from Michał K.) and <a href=http://www.emdbiosciences.com/docs/docs/PROT/TB045.pdf>pET15b</a>.</li>
 +
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htmtop10">TOP10</a> strain with ligation products. </li>
 +
<li> Transformants plating on LB + ampicillin.</li></ol>
 +
</p>
 +
</html>

Latest revision as of 14:05, 28 October 2008

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
Interactive list of topics
next notebook entry

 


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

Piotr, Weronika, Emilia

  1. Gel electrophoresis and gel-out of proper bands.
  2. Ligation of pZC320 with standard parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
  3. Chemotransformation of E.coli TOP10 with ligation products.
  4. Plating transformants on LB+Amp30+X-gal+IPTG.

Preparation of constructs on pET15b: OmpA_alpha and OmpA_omega

Paweł

  1. Clean-up of overnight digest.
  2. Ligation of OmpA_alpha (from Michał K.) and pET15b.
  3. Ligation of OmpA_omega (from Michał K.) and pET15b.
  4. Transformation of E. coli TOP10 strain with ligation products.
  5. Transformants plating on LB + ampicillin.

Retrieved from "http://2008.igem.org/Team:Warsaw/Calendar-Main/2_July_2008"