Team:Warsaw/Calendar-Main/10 October 2008
From 2008.igem.org
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<h3>Visit in US Embassy</h3> | <h3>Visit in US Embassy</h3> | ||
+ | <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>ΔA (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>ΔA (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> | ||
- | <h3>Preparation of | + | <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 | + | <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> | + | <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> | + | <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# | + | <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 °C; elongation length 2.30 min) to | + | primers (annealing temperature 40 - 60 °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 °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> | + | <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# | + | <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 °C; elongation length 2.30 min) to obtain | + | primers (annealing temperature 45 °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°C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 60°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 | + | |
+ | <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+ | + | <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_ΔA_alpha</a> plasmid using |
- | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+ | + | <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 °C; elongation length 60s) to obtain alpha fragment. </li> | primers (annealing temperature 58 °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
Visit in US EmbassyVisas have been accorded to the whole team. Preparation of alpha_linker under PT7 (BBa_K103019)Michał K., Piotr
Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha Preparation of omega_linker under PT7 (BBa_K103020)Michał K., Piotr
Preparation of AID under pBAD/araC (BBa_K103002)Michał K., Piotr
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 Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)Michał K.
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
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