Team:Warsaw/Calendar-Main/18 September 2008
From 2008.igem.org
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+ | <h3>Preparation of pACYC177 + OmpA_omega_</h3> | ||
+ | <h4>Michał K.</h4> | ||
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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 | ||
+ | <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 OmpA_omega fragment. </li> | ||
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+ | <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_omega - 900 bp).</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> | ||
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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 products.</li></ol> | ||
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<h3>Preparation of BioBricks</h3> | <h3>Preparation of BioBricks</h3> | ||
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<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/Team:Warsaw/Calendar-Main/18_September_2008#fig4">Fig. 4</a>. <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/Team:Warsaw/Calendar-Main/18_September_2008#fig4">Fig. 4</a>. <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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<li> Gel electrophoresis of digested plasmids | <li> Gel electrophoresis of digested plasmids |
Revision as of 23:03, 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 pACYC177 + OmpA_omega_Michał 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 Fig. 3. PCR to obtain linker_alpha 1. Marker 2. linker_alpha Fig. 4. Control SacI/NdeI digest of pET15b_OmpA_omega 1. Marker 2. digested pSB1A3 Fig. 5. Control EcoRI/BcuI digest of pSB1A3 1. Marker 2. digested pSB1A3
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