Team:Warsaw/Calendar-Main/18 September 2008
From 2008.igem.org
(Difference between revisions)
MKrzyszton (Talk | contribs) |
|||
(27 intermediate revisions not shown) | |||
Line 22: | Line 22: | ||
primers (elongation length 60s) to obtain <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103009>linker_alpha (BBa_K103009)</a> fragment. Reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).</li> | primers (elongation length 60s) to obtain <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103009>linker_alpha (BBa_K103009)</a> fragment. Reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).</li> | ||
- | <li> Gel electrophoresis of PCR products <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig1">Fig. 1</a>.</li></ol> | + | <li> Gel electrophoresis of PCR products (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig1">Fig. 1.</a>).</li></ol> |
- | < | + | |
+ | <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> | ||
+ | 2. 55 °C linker_alpha<br> | ||
+ | 3. 60 °C linker_alpha<br> | ||
+ | 4. 65 °C linker_alpha<br> | ||
+ | 5. 70 °C linker_alpha<br></var> | ||
+ | |||
+ | |||
+ | |||
+ | <h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>ΔA (BBa_K103003)</a></h3> | ||
<h4>Michał K.</h4> | <h4>Michał K.</h4> | ||
<ol> | <ol> | ||
Line 32: | Line 42: | ||
primers (annealing temperature 58 °C; elongation length 45s) to obtain ΔA fragment. </li> | primers (annealing temperature 58 °C; elongation length 45s) to obtain ΔA fragment. </li> | ||
- | <li> Gel electrophoresis | + | <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 (ΔA - 250 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig2">Fig. 2</a>.</li> |
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product and <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> standard plasmid with EcoRI and BcuI (BamHI buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li> | <li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product and <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> standard plasmid with EcoRI and BcuI (BamHI buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</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> | ||
- | <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> |
+ | |||
+ | |||
+ | <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> | ||
+ | |||
+ | |||
- | <h3>Preparation of | + | <h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a></h3> |
<h4>Michał K.</h4> | <h4>Michał K.</h4> | ||
<ol> | <ol> | ||
Line 46: | Line 70: | ||
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="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> | ||
+ | 2. linker_alpha<br></var> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a></h3> | ||
<h4>Michał K.</h4> | <h4>Michał K.</h4> | ||
<ol> | <ol> | ||
Line 58: | Line 91: | ||
primers (annealing temperature 55 °C; elongation length 60s) to obtain <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a> fragment. </li> | primers (annealing temperature 55 °C; elongation length 60s) to obtain <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</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 band (linker_omega - 350 bp).</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 band (linker_omega - 350 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig2">Fig. 2</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> | ||
+ | |||
+ | <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> | ||
+ | |||
</ol> | </ol> | ||
- | <h3>Preparation of | + | <h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a></h3> |
<h4>Michał K.</h4> | <h4>Michał K.</h4> | ||
Line 71: | Line 113: | ||
<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).</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><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> | ||
+ | 1. Marker<br> | ||
+ | 2. deltaA<br> | ||
+ | 3. omega<br> | ||
+ | 4. ompA_omega<br></var> | ||
Line 86: | Line 134: | ||
- | <h3>Preparation of <a href=http://partsregistry.org/ | + | <h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a></h3> |
<h4>Michał K.</h4> | <h4>Michał K.</h4> | ||
Line 104: | Line 152: | ||
<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 ( | + | 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> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
</html> | </html> |
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
|