Team:Warsaw/Calendar-Main/18 September 2008
From 2008.igem.org
(Difference between revisions)
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<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 | + | 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> |
<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> | ||
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<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 | + | primers (annealing temperature 65 °C; elongation length 60s) to obtain linker_alpha 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 ( | + | <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><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> | ||
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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">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 | + | primers (annealing temperature 55 °C; elongation length 60s) to obtain linker_omega 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 ( | + | <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><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> | ||
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primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA_omega fragment. </li> | primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA_omega 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 (ΔA - 250 bp, | + | <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 (ΔA - 250 bp, linker_alpha - 600 bp, linker_omega - 350 bp and OmpA_omega - 900 bp).</li> |
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR products and <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> standard plasmid with EcoRI and BcuI (BamHI buffer) and <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 plasmids.</li> | <li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR products and <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> standard plasmid with EcoRI and BcuI (BamHI buffer) and <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 plasmids.</li> | ||
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/c/c3/Grad2_17_09.jpg" width=300/></a><var><b>Fig. 1. Gradient PCR (temperatures:55-75 °C)</b><br> | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/c/c3/Grad2_17_09.jpg" width=300/></a><var><b>Fig. 1. Gradient PCR (temperatures:55-75 °C)</b><br> | ||
1. Marker<br> | 1. Marker<br> | ||
- | 2. 55 °C | + | 2. 55 °C linker_alpha<br> |
- | 3. 60 °C | + | 3. 60 °C linker_alpha<br> |
- | 4. 65 °C | + | 4. 65 °C linker_alpha<br> |
- | 5. 70 °C | + | 5. 70 °C linker_alpha<br></var> |
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d0/Go_17_09.jpg" width=300/></a><var><b>Fig. 2. PCR to obtain inserts</b><br> | <a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d0/Go_17_09.jpg" width=300/></a><var><b>Fig. 2. PCR to obtain inserts</b><br> |
Revision as of 22:35, 26 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.
Preparation of pSB1A3 carrying ΔA (BBa_K103003)Michał K.
Preparation of pSB2K3 + _alphaMichał K.
Preparation of pSB2K3 + _omegaMichał K.
Preparation of BioBricksMichał K.
1. Marker 2. 55 °C linker_alpha 3. 60 °C linker_alpha 4. 65 °C linker_alpha 5. 70 °C linker_alpha Fig. 2. PCR to obtain inserts 1. Marker 2. deltaA 3 omega 4. ompA_omega
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