Team:Warsaw/Calendar-Main/17 September 2008

From 2008.igem.org

(Difference between revisions)
 
(27 intermediate revisions not shown)
Line 1: Line 1:
{{WarNotebook}}
{{WarNotebook}}
<!-- do not edit above me! -->
<!-- do not edit above me! -->
 +
<html>
 +
<h3>'Hunter and prey' system tests: Competition tests</h3>
 +
<h4>Piotr</h4>
 +
<p><ol><li>Plasmid <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>isolation</a> from previous day's cultures.</li>
 +
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> with SacI and BamHI.</li><li>Electrophoresis (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/17_September_2008#fig1">Fig. 1</a>).</li></ol></p>
 +
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/9/98/Konkurencja1.jpg" width=180/></a>
 +
<var><b>Fig. 1.</b> Result of competition test:<br>
 +
1 and 5 - DNA ladder,<br>
 +
2 - Insert from isolated plasmid refers to OmpA_A_Alpha <br>(Z_Omega protein added to the medium),<br>
 +
3 - Insert from isolated plasmid refers to OmpA_A_Omega <br>(Z_Alpha protein added to the medium),<br>
 +
4 - Insert from isolated plasmid refers to OmpA_Z_Omega <br>(A_Alpha protein added to the medium).</var>
 +
 +
<p>Conclusion: cells with interacting protein survive competition!</p>
 +
 +
<h3>MutD5 testing</h3>
 +
<h4>Emilia</h4>
 +
<p>Inoculation of MutD5 into medium with tetracycline.</p>
 +
 +
<h3>Optimisation of primers for preparation of parts</h3>
 +
 +
<h4>Michał K.</h4>
 +
<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using
 +
 +
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL_BNXNE">AL_BNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>
 +
 +
primers (elongation length 45s) to obtain &Delta;A fragment. </li>
 +
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using
 +
 +
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlfaPSpe">AlfaPSpe</a>
 +
primers (elongation length 60s) to obtain link_alpha fragment. </li>
 +
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a> plasmid using
 +
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegPSpe">OmegPSpe</a>
 +
primers (elongation length 60s) to obtain link_omega fragment. </li>
 +
 +
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using
 +
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpLNXNE">OmpLNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
 +
primers (elongation length 90s) to obtain OmpA_omega fragment. </li>
 +
<p>Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 &deg;C (four reactions).</p>
 +
<li> Gel electrophoresis of PCR products (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/17_September_2008#fig2">Fig. 2.</a>).</li>
 +
</ol>
 +
 +
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/9/9e/Grad_17_09.jpg" width=300/></a><var><b>Fig. 2.</b> Gradient PCR (temperatures:55-75 &deg;C)<br>
 +
1. Marker<br>
 +
2. 55 &deg;C deltaA<br>
 +
3. 60 &deg;C deltaA<br>
 +
4. 65 &deg;C deltaA<br>
 +
5. 70 &deg;C deltaA<br>
 +
6. 55 &deg;C link_omega<br>
 +
7. 60 &deg;C link_omega<br>
 +
8. 65 &deg;C link_omega<br>
 +
9. 70 &deg;C link_omega<br>
 +
10. 55 &deg;C link_alpha<br>
 +
11. 60 &deg;C link_alpha<br>
 +
12. 65 &deg;C link_alpha<br>
 +
13. 70 &deg;C link_alpha<br>
 +
14. 55 &deg;C OmpA_omega<br>
 +
15. 60 &deg;C OmpA_omega<br>
 +
16. 65 &deg;C OmpA_omega<br>
 +
17. 70 &deg;C OmpA_omega<br></var>
-
<!-- Zaszczepienie mamutów do 100ml pożywki i chemokompetenty z nich
 
-
strzelenie do nich rużnymi wariantami ompów (też a zmutowane) -->
 
-
<h4>Piotr</h4>
 
-
<p>Inoculation of mutD5 into 100 ml of LB and rendering them chemocompetent. Electroporation with various OmpA variants. </p>
 
 +
</html>
<!-- do not remove this! -->
<!-- do not remove this! -->
{{WarNotebookEnd}}
{{WarNotebookEnd}}

Latest revision as of 15:15, 29 October 2008

Gallery Bricks Notebook Team Project Home


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

 


'Hunter and prey' system tests: Competition tests

Piotr

  1. Plasmid isolation from previous day's cultures.
  2. Digest with SacI and BamHI.
  3. Electrophoresis (Fig. 1).

Fig. 1. Result of competition test:
1 and 5 - DNA ladder,
2 - Insert from isolated plasmid refers to OmpA_A_Alpha
(Z_Omega protein added to the medium),
3 - Insert from isolated plasmid refers to OmpA_A_Omega
(Z_Alpha protein added to the medium),
4 - Insert from isolated plasmid refers to OmpA_Z_Omega
(A_Alpha protein added to the medium).

Conclusion: cells with interacting protein survive competition!

MutD5 testing

Emilia

Inoculation of MutD5 into medium with tetracycline.

Optimisation of primers for preparation of parts

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AL_BNXNE and APSacSpe primers (elongation length 45s) to obtain ΔA fragment.
  2. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using LinLSXNE and AlfaPSpe primers (elongation length 60s) to obtain link_alpha fragment.
  3. PCR on pACYC177 + OmpA_omega plasmid using LinLSXNE and OmegPSpe primers (elongation length 60s) to obtain link_omega fragment.
  4. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmpLNXNE and LinP_BS primers (elongation length 90s) to obtain OmpA_omega fragment.

    5. Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).

  5. Gel electrophoresis of PCR products (Fig. 2.).
Fig. 2. Gradient PCR (temperatures:55-75 °C)
1. Marker
2. 55 °C deltaA
3. 60 °C deltaA
4. 65 °C deltaA
5. 70 °C deltaA
6. 55 °C link_omega
7. 60 °C link_omega
8. 65 °C link_omega
9. 70 °C link_omega
10. 55 °C link_alpha
11. 60 °C link_alpha
12. 65 °C link_alpha
13. 70 °C link_alpha
14. 55 °C OmpA_omega
15. 60 °C OmpA_omega
16. 65 °C OmpA_omega
17. 70 °C OmpA_omega


Retrieved from "http://2008.igem.org/Team:Warsaw/Calendar-Main/17_September_2008"