|
|
| (16 intermediate revisions not shown) |
| Line 4: |
Line 4: |
| | | | |
| | <html> | | <html> |
| - | <ol><li>Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_A from previous day) inoculated to liquid LB with kanamycin.
| |
| - | </li></ol>
| |
| | | | |
| | | | |
| - | <h4>Antoni:</h4> | + | <h3>Cloning of protein Z DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-omega</a> in place of OmpA</h3><h4>Paweł</h4> |
| | + | <ol><li>Results of ligation: 6 colonies grown.</li> |
| | + | <li>Each colony cultured overnight in LB + ampicillin.</li></ol> |
| | | | |
| - | < ol> <li>Preparation of < a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/ protocols#chemocompetent"> chemocompetent</ a> bacteria < i> E. coli< /i> <a href= "https://2008.igem.org/Wiki/Team:Warsaw/ igem_notebook.htmtop10"> TOP10</a>. </li></ol> | + | <h3>Cloning of protein A DNA to OmpA constructs</h3><h4>Michał K.</h4> |
| | + | <ol> |
| | + | < li> Colony PCR on colonies from plates with transformations <a href=https://2008.igem.org/Wiki/Team:Warsaw/ vectors/pACYC177%2BompA-A-omega> pACYC177+OmpA_A_omega</a>. Primers used: |
| | | | |
| - | <h3>Cloning omega-A fusion on pKS (second attempt)</h3> | + | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a>.</li><li> Gel electrophoresis.</li> |
| - | <h4>Michał L., Ewa, Marcin:</h4> | + | <li>Confirmed transformant colonies inoculated to liquid LB with kanamycin. </li></ol> |
| - | <p>Sequencing confirms that we have obtained proper construct with omega-A fusion</p> | + | |
| | | | |
| | | | |
| | | | |
| | + | <h3>Preparation of chemocompetent bacteria</h3> |
| | + | <h4>Antoni</h4> |
| | | | |
| - | | + | < p> Preparation of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols# chemocompetent"> chemocompetent</a> bacteria < i> E. coli</ i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook. htmtop10">TOP10</a>.</p> |
| - | | + | |
| - | <h3>Cloning of protein A DNA to OmpA constructs</h3> | + | |
| - | <ol><li>Colony PCR on colonies from plates with transformations pACYC177+OmpA_A_omega. Primers used:
| + | |
| - | | + | |
| - | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a></li>
| + | |
| - | <li>Confirmed transformant colonies inoculated to liquid LB with kanamycin. </li>
| + | |
| - | <li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols# plasmid_DNA_isolation"> Isolation of plasmids</a> from cultures inocluated on previous day (pACYC177+OmpA_A_alpha). < /li> | + | |
| - | <li>Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with BamHI and SacI.</li></ol>
| + | |
| - | </p>
| + | |
| - | <h3>Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA<br>
| + | |
| - | Paweł</h3>
| + | |
| - | <ol><li> Ligations transformed into <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook. htm#top10">TOP10</a> and plated on LB + ampicillin. </li></ol> | + | |
| - | | + | |
| - | <h3>Cloning omega-A fusion on pKS (second attempt)</h3>
| + | |
| - | <h4>Michał L., Ewa, Marcin:</h4>
| + | |
| - | <ol>
| + | |
| - | <li>We have obtained white colonies of transformants and inoculated 10 of them on liquid LB+Amp<sub>100</sub></li>
| + | |
| - | | + | |
| - | <h3>Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA<br>
| + | |
| - | Paweł</h3>
| + | |
| - | <p><ol>
| + | |
| - | <li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmids from cultures inocluated on previous day</li>
| + | |
| - | <li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with NdeI and NotI. Two positives obtained (proper band: ~150 bp visible)</li>
| + | |
| - | <li>One of positive plasmids transformed into <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> and plated on LB+amp, overnight, for further isolation of pET15b+Z+omega vector</li>
| + | |
| - | </ol></p>
| + | |
| | | | |
| | | | |
"
