Team:Warsaw/Calendar-Main/10 October 2008

From 2008.igem.org

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<html>
<h3>Visit in US Embassy</h3>
<h3>Visit in US Embassy</h3>
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<h4></h4>
<p>Visas have been accorded to the whole team.</p>
<p>Visas have been accorded to the whole team.</p>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
<A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> with EcoRI and SacI (BamHI buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> with CIAP </li>
<A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> with EcoRI and SacI (BamHI buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> with CIAP </li>
-
<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 2200 bp. </li>
+
<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 2200 bp. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig1">Fig. 1</a>. </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested  <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> with <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a> fragment (1 hr).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested  <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> with <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a> fragment (1 hr).</li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with above ligation.</li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with above ligation.</li>
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</ol>
</ol>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a></h3>
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<p class="hide"><img src="https://static.igem.org/mediawiki/2008/f/fd/Go_10_10_2008.jpg">
 +
<var><b>Fig. 1.</b>PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha<br>
 +
1. Marker<br>
 +
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)<br>
 +
3. PCR to obtain AID under pBAD/araC (BBa_K103002)<br> 
 +
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha<br></var></p>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a></h3>
<h4>Michał K., Piotr</h4>
<h4>Michał K., Piotr</h4>
<ol>
<ol>
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<h3>Preparation of AraC+pBAD+AID</h3>
+
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a></h3>
<h4>Michał K., Piotr</h4>
<h4>Michał K., Piotr</h4>
<ol>
<ol>
-
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day - <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> without EcoRI site.</li>
+
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day - <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site).</li>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.</li>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.</li>
-
<li>Temperature gradient <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/pMPM-T5-AID>pMPMT5+AID</a> without EcoRI site plasmid using  
+
<li>Temperature gradient <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/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site) plasmid using  
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AraCl">AraCl</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDP_HindSpeNotPst">AIDP_HindSpeNotPst</a>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AraCl">AraCl</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpHSNP">AIDP_HindSpeNotPst</a>
-
  primers (annealing temperature 40 - 60 &deg;C; elongation length 2.30 min) to obtain AraC+pBAD+AID fragment. Gel electrophoresis.</li>
+
  primers (annealing temperature 40 - 60 &deg;C; elongation length 2.30 min) to optimize conditions for obtaining <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a>. </li>
 +
<li>Gel electrophoresis. Best annealing temperature (45 &deg;C) chosen. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig2">Fig. 2</a>.</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/pMPM-T5-AID>pMPMT5+AID</a> without EcoRI site plasmid using  
+
<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/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site) plasmid using  
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AraCl">AraCl</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDP_HindSpeNotPst">AIDP_HindSpeNotPst</a>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AraCl">AraCl</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpHSNP">AIDP_HindSpeNotPst</a>
-
  primers (annealing temperature 45 &deg;C; elongation length 2.30 min) to obtain AraC+pBAD+AID fragment. </li>
+
  primers (annealing temperature 45 &deg;C; elongation length 2.30 min) to obtain <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a>fragment. </li>
-
<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band 1800 bp. </li>
+
<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band 1800 bp. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig1">Fig. 1</a>. </li>
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of  
isolated PCR product with XbaI and PstI (Tango buffer). </li>
isolated PCR product with XbaI and PstI (Tango buffer). </li>
<li>  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product. </li></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 digested PCR product. </li></ol>
 +
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/f/fe/Grad_arac_09_10_2008.jpg"></a>
 +
<var><b>Fig. 2.</b>Gradient PCR products for AID under pBAD/araC<br> 1 -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 40&deg;C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 60&deg;C (the highest temperature of gradient).</var>
 +
<p class="hide"><img src="https://static.igem.org/mediawiki/2008/f/fd/Go_10_10_2008.jpg">
 +
<var><b>Fig. 1.</b>PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha<br>
 +
1. Marker<br>
 +
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)<br>
 +
3. PCR to obtain AID under pBAD/araC (BBa_K103002)<br> 
 +
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha<br></var></p>
-
<h3>Preparation of pLac_OmpA_alpha</h3>
+
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103017>OmpA_linker_alpha_linker under Plac (BBa_K103017)</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol>
<ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using  
+
<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#AlphaL+Nde">AlphaL+Nde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP+link10+homo2">AlphaP+link10+homo2</a>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP+link10+homo2">AlphaP+link10+homo2</a>
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha fragment. </li>
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha fragment. </li>
-
<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band 600 bp. </li>
+
<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band 600 bp. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig1">Fig. 1</a>. </li>
</ol>
</ol>
-
 
+
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/f/fd/Go_10_10_2008.jpg"></a>
 +
<var><b>Fig. 1.</b>PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha<br>
 +
1. Marker<br>
 +
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)<br>
 +
3. PCR to obtain AID under pBAD/araC (BBa_K103002)<br> 
 +
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha<br></var>
</html>
</html>

Latest revision as of 12:26, 29 October 2008

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Visit in US Embassy

Visas have been accorded to the whole team.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K., Piotr

  1. Clean-up of overnight digest reaction.
  2. Digest of pSB1A3 carrying ΔA (BBa_K103003) with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP
  3. Gel electrophoresis and gel-out of proper band - 2200 bp. Fig. 1.
  4. Ligation of digested pSB1A3 with alpha_linker under PT7 (BBa_K103019) fragment (1 hr).
  5. Transformation of TOP10 with above ligation.
  6. Tranformants plating on LB with ampicillin.

Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K., Piotr

  1. Ligation of digested pSB2K3 vector from (30 September) with omega_linker under PT7 (BBa_K103020) fragment (1 hr).
  2. Transformation of TOP10 with above ligation.
  3. Tranformants plating on LB with kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day - pMPMT5+AID (with removed EcoRI site).
  2. Control digest of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.
  3. Temperature gradient PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 40 - 60 °C; elongation length 2.30 min) to optimize conditions for obtaining AID under pBAD/araC (BBa_K103002).
  4. Gel electrophoresis. Best annealing temperature (45 °C) chosen. Fig. 2.
  5. PCR on pMPMT5+AID (with removed EcoRI site) plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 45 °C; elongation length 2.30 min) to obtain AID under pBAD/araC (BBa_K103002)fragment.
  6. Gel electrophoresis and gel-out of proper band 1800 bp. Fig. 1.
  7. Digest of isolated PCR product with XbaI and PstI (Tango buffer).
  8. Clean-up of digested PCR product.
Fig. 2.Gradient PCR products for AID under pBAD/araC
1 -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 40°C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 60°C (the highest temperature of gradient).

Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AlphaL+SacI and AlphaP+link10+homo2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha fragment.
  2. Gel electrophoresis and gel-out of proper band 600 bp. Fig. 1.
Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha
1. Marker
2. PCR to obtain alpha_linker under PT7 (BBa_K103019)
3. PCR to obtain AID under pBAD/araC (BBa_K103002)
4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha