|
|
| (14 intermediate revisions not shown) |
| Line 3: |
Line 3: |
| | | | |
| | <html> | | <html> |
| - | <h3>Purification of proteins: A-alpha, Z-alpha and Z-omega<br> | + | <h3>Purification of proteins: A-alpha</h3><h4>Piotr, Emilia</h4> |
| - | Piotr </h3> | + | <p><ol><li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BA-alpha>pET15b+A_alpha</a> in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain from overnight culture inoculated in fresh LB and cultured until OD=0.5.</li> |
| - | <p>A-alpha, Z-alpha and Z-omega from overnight culture were inoculated in fresh LB, cultured until OD=0,5 and then induced with different concentrations of IPTG in 22C and 37C. Samples were collected twice: after 3h and next day (samples were centrifuged and frozen)</p> | + | <li>Culture was induced with different concentrations of IPTG at 22°C and 37°C.</li> |
| | + | <li>Samples were collected twice: after 3h and next day (samples were centrifuged and frozen).</li></ol></p> |
| | | | |
| - | <h3>Mutagenesis of protein A<br>Paweł</h3>
| |
| - | <p>Mutagenesis of protein A was performed using 2 pairs of primers: <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelL+KpnI>ADelL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelP>ADelP</a> (deletion of aminoacids involved in interaction with protein Z) and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutL+KpnI>AMutL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutP>AMutP</a> (changing of few aminoacids involved in interaction with protein Z).</p><br>
| |
| - | <p>Mutagenesis were performed on 3 vectors: pACYClac+ompA-A-omega, pACYClac+ompa-A-alfa and pACYClac+ ompa-alfa-A.</p>
| |
| - | <p>Each mutagenesis were performed using each primer at final concentration 0,1 uM, dNTPs at final concentration 0,25 uM and Walk (Pwo) polymerase (shipped by <a href=http://aabiot.com/>A&A Biotechnology</a>), with 100 ng of DNA template. </p>
| |
| - | <p><br>PCR program (15 cycles):
| |
| - | <pre style="text-align: left">94C 5 min
| |
| | | | |
| - | 94C 30 s
| |
| - | 55C 30 s
| |
| - | 72C 10 min
| |
| - |
| |
| - | 72C 8 min
| |
| - |
| |
| - | 4C pause
| |
| - | </pre></p>
| |
| | </html> | | </html> |
| | <!-- do not remove this! --> | | <!-- do not remove this! --> |
| | {{WarNotebookEnd}} | | {{WarNotebookEnd}} |
"
