Team:Warsaw/Calendar-Main/23 September 2008

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Revision as of 21:22, 21 October 2008


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PNK phosporylation of mutagenesis products for 30 min at 37°C.
PNK heat-inactivated by incubation at 75°C for 15 min.
T4 ligase added and incubated for 1 h at room temperature.
T4 ligase inactivated by incubation at 55°C for 20 min, then 5U of DpnI added and incubated at 37°C for 3 hours.
After DpnI treatment mutagenesis products transformed into TOP10 and plated on LB + kanamycin plates.

Isolation of plasmids from cultures inoculated on previous day.

Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). No proper colonies found.

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 <li>T4 ligase added and incubated for 1 h at room temperature.</li>
 <li>T4 ligase inactivated by incubation at 55&deg;C for 20 min, then 5U of DpnI added and incubated at 37&deg;C for 3 hours.</li> <li>After DpnI treatment mutagenesis products transformed into <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> and plated on LB + kanamycin plates.</li></ol></p>
+<h3>Preparation of BioBricks</h3>
+<h4>Michał K.</h4>
+<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmids from cultures inoculated 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 EcoRI and PstI (Orange buffer). No proper colonies found.</li>
+</ol></p>
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