Team:Warsaw/Calendar-Main/18 September 2008
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- | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/c/c3/Grad2_17_09.jpg" width= | + | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/c/c3/Grad2_17_09.jpg" width=180/></a><var><b>Fig. 1. </b>Gradient PCR (temperatures:55-75 °C)<br> |
1. Marker<br> | 1. Marker<br> | ||
2. 55 °C linker_alpha<br> | 2. 55 °C linker_alpha<br> | ||
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<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> | <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> | ||
- | <li> Gel electrophoresis of digested plasmid and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (<a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> - 2200 bp).</li></ol> | + | <li> Gel electrophoresis of digested plasmid and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (<a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> - 2200 bp).<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig3">Fig. 3</a>.</li></ol> |
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+ | <p class="hide"><br> | ||
+ | <img src="https://static.igem.org/mediawiki/2008/d/d0/Go_17_09.jpg" width="300"/><var><b>Fig. 2.</b> PCR to obtain inserts<br> | ||
+ | 1. Marker<br> | ||
+ | 2. DeltaA<br> | ||
+ | 3. Omega<br> | ||
+ | 4. OmpA_omega<br></var> | ||
+ | </p> | ||
+ | <a name="fig3"><img src="https://static.igem.org/mediawiki/2008/9/96/Go2_19_09.jpg" width=180/></a><var><b>Fig. 3.</b> EcoRI/BcuI digest of pSB1A3 <br> | ||
+ | 1. Marker<br> | ||
+ | 2. Digested pSB1A3<br></var> | ||
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+ | |||
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a></h3> | <h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a></h3> | ||
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primers (annealing temperature 65 °C; elongation length 60s) to obtain <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103009>linker_alpha (BBa_K103009) fragment. </li> | primers (annealing temperature 65 °C; elongation length 60s) to obtain <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103009>linker_alpha (BBa_K103009) fragment. </li> | ||
- | <li> Gel electrophoresis of PCR product | + | <li> Gel electrophoresis of PCR product and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (linker_alpha - 600 bp).<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig4">Fig. 4</a>.</li> |
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with EcoRI and BcuI (BamHI buffer). </li> | <li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with EcoRI and BcuI (BamHI 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> | <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> | </ol> | ||
- | + | ||
- | <a name=" | + | <a name="fig4"><img src="https://static.igem.org/mediawiki/2008/8/85/Go_1909_2008.jpg" width=180/></a><var><b>Fig. 4.</b> PCR to obtain linker_alpha for reisolation from agarose gel<br> |
1. Marker<br> | 1. Marker<br> | ||
- | 2. | + | 2. linker_alpha<br></var> |
- | + | ||
- | + | ||
- | + | ||
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<p class="hide"><br> | <p class="hide"><br> | ||
- | + | <img src="https://static.igem.org/mediawiki/2008/d/d0/Go_17_09.jpg" width="300"/><var><b>Fig. 2.</b> PCR to obtain inserts<br> | |
1. Marker<br> | 1. Marker<br> | ||
- | 2. | + | 2. DeltaA<br> |
- | 3 | + | 3. Omega<br> |
- | 4. | + | 4. OmpA_omega<br></var> |
</p> | </p> | ||
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<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 | <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#OmpLNXNE">OmpLNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> | <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 (annealing temperature 58 °C; elongation length 90s) to obtain <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> fragment. </li> | + | primers (annealing temperature 58 °C; elongation length 90s) to obtain <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> fragment.</li> |
<li> Gel electrophoresis of PCR products and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (OmpA-linker-omega-linker - 900 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig2">Fig. 2</a>.</li> | <li> Gel electrophoresis of PCR products and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (OmpA-linker-omega-linker - 900 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig2">Fig. 2</a>.</li> | ||
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with EcoRI and BcuI (BamHI buffer).</li> | <li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with EcoRI and BcuI (BamHI 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 products.</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 products.</li></ol><br> |
+ | Mentioned primers were used only for test purposes. During part preparation we've used another primers. | ||
- | + | <a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d0/Go_17_09.jpg" width=220/></a><var><b>Fig. 2.</b> PCR products for reisolation from agarose gel<br> | |
- | <a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d0/Go_17_09.jpg" width= | + | |
1. Marker<br> | 1. Marker<br> | ||
2. deltaA<br> | 2. deltaA<br> | ||
- | 3 omega<br> | + | 3. omega<br> |
4. ompA_omega<br></var> | 4. ompA_omega<br></var> | ||
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<li> Gel electrophoresis of digested plasmid | <li> Gel electrophoresis of digested plasmid | ||
- | and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (OmpA_linker - 500 bp) | + | and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (OmpA_linker - 500 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig5">Fig. 5</a>.</li> |
+ | <a name="fig5"><img src="https://static.igem.org/mediawiki/2008/6/6a/Go_29_09.jpg" width=180/></a><var><b>Fig. 5. </b>SacI/NdeI digest of pET15b_OmpA_omega <br> | ||
+ | 1. Marker<br> | ||
+ | 2. digested pSB1A3<br></var> | ||
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Latest revision as of 22:16, 28 October 2008
'Hunter and prey' system tests: Competition testsWeronikaInoculation of pACYC177+OmpA_A_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_alpha + induction using 0.25mM iPTG MutD5 testingPiotrInoculation of mutD5 into 100 ml of LB and rendering them chemocompetent. Electroporation with pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_omega_ΔA. Optimisation of primers for preparation of partsMichał K.
1. Marker 2. 55 °C linker_alpha 3. 60 °C linker_alpha 4. 65 °C linker_alpha 5. 70 °C linker_alpha Preparation of ΔA (BBa_K103003)Michał K.
1. Marker 2. Digested pSB1A3 Preparation of linker_alpha (BBa_K103009)Michał K.
1. Marker 2. linker_alpha Preparation of linker_omega (BBa_K103013)Michał K.
Preparation of OmpA-linker-omega-linker (BBa_K103016)Michał K.
Mentioned primers were used only for test purposes. During part preparation we've used another primers. Fig. 2. PCR products for reisolation from agarose gel 1. Marker 2. deltaA 3. omega 4. ompA_omega Preparation of OmpA-linker (BBa_K103006)Michał K.
1. Marker 2. digested pSB1A3
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