Team:Warsaw/Calendar-Main/18 September 2008
From 2008.igem.org
(Difference between revisions)
(38 intermediate revisions not shown) | |||
Line 20: | Line 20: | ||
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPSpe">AlphaPSpe</a> | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPSpe">AlphaPSpe</a> | ||
- | primers (elongation length 60s) to obtain linker_alpha 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> |
- | <h3>Preparation of <a href=http://partsregistry.org/Part: | + | |
+ | <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 <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 44: | Line 68: | ||
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPSpe">AlphaPSpe</a> | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPSpe">AlphaPSpe</a> | ||
- | primers (annealing temperature 65 °C; elongation length 60s) to obtain linker_alpha 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 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).</li> | + | <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> | ||
- | <h3>Preparation of <a href=http://partsregistry.org/Part: | + | |
+ | <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> | ||
<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 | <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> | <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 (annealing temperature 55 °C; elongation length 60s) to obtain linker_omega 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> | ||
+ | |||
+ | <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_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> | ||
+ | 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><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><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> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <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 77: | Line 146: | ||
- | |||
- | |||
- | |||
- | + | <li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA_alpha</a> plasmid with NdeI and SacI (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 | + | |
- | |||
- | |||
- | < | + | <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). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig5">Fig. 5</a>.</li> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | <a name=" | + | <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> | 1. Marker<br> | ||
- | 2. | + | 2. digested pSB1A3<br></var> |
- | + | ||
- | + | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
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. ![]() 1. Marker 2. deltaA 3. omega 4. ompA_omega Preparation of OmpA-linker (BBa_K103006)Michał K.
![]() 1. Marker 2. digested pSB1A3
|
![]() |