|
|
| Line 2: |
Line 2: |
| | <!-- do not edit above me! --> | | <!-- do not edit above me! --> |
| | | | |
| - | <html>
| + | emptyday |
| - | <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. Zero positive results obtained - just empty vectors</li>
| + | |
| - | <li>Another overnight <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation>ligation</a> of pET15b-OmpA-omega (NdeI/NotI cutted) with protein Z DNA</li>
| + | |
| - | </ol></p>
| + | |
| - | | + | |
| - | <h3>Cloning omega-A fusion on pKS (second attempt)</h3>
| + | |
| - | <h4>Michał L., Ewa, Marcin:</h4>
| + | |
| - | <ol>
| + | |
| - | <li>Since the phage is gone we can continue this cloning:</li>
| + | |
| - | <li>Restriction digest of PCL product and pKS vector with SacI and NotI</li>
| + | |
| - | <li>Ligation of omega-A fusion with pKS vector</li>
| + | |
| - | <li>Transformation of Top10 with ligation product</li>
| + | |
| - | | + | |
| - | </ol>
| + | |
| - | | + | |
| - | <h3>Cloning of protein A DNA to OmpA constructs</h3>
| + | |
| - | <p><ol>
| + | |
| - | <li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (pACYC177 were also dephosporylated with CIAP)</li>
| + | |
| - | <li> Gel electrophoresis 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 (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane). </li>
| + | |
| - | <li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of A DNA fragment with both pACYC177 vectors.</li>
| + | |
| - | <li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligations. </li>
| + | |
| - | <li>Transformants plating on LB + kanamycin. </li></ol>
| + | |
| - | </p>
| + | |
| - | <h3>Cloning of protein Z DNA to OmpA constructs<br>Michał K.</h3>
| + | |
| - | <p><ol>
| + | |
| - | <li> Isolation of plasmids from cultures inocluated on previous day (pACYC177+OmpA_Z_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 (we found 4 good clones).</li></ol></p>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | </html>
| + | |
| - | | + | |
| | | | |
| | <!-- do not remove this! --> | | <!-- do not remove this! --> |
| | {{WarNotebookEnd}} | | {{WarNotebookEnd}} |
"
