Team:Warsaw/Calendar-Main/12 August 2009
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<p>Task 1:</p> | <p>Task 1:</p> | ||
<ul> | <ul> | ||
- | <li>Digest of isolate plasmids | + | <li>Digest of isolate plasmids to verify the success of isolation</li> |
</ul> | </ul> | ||
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
- | <li>Samples containing plasmids digested in 11.08.2009 were thawed and loaded into the agarose gel</li> | + | <li>Samples containing plasmids digested in <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/11_August_2009">11.08.2009</a> were thawed and loaded into the agarose gel</li> |
</ul> | </ul> | ||
<p><b>Comment:</b></p> | <p><b>Comment:</b></p> | ||
+ | <p>Results:</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2009/8/87/R0080_E0022_cro_digest_12_08_09.png" widht="40%" height="40%"> | ||
+ | <font face="Times New Roman" size="3"><p><div style="text-align: center;">verification of the restriction patterns</div></p></font> | ||
+ | <p><b>Comment</b></p> | ||
<p>All isolation were successful</p> | <p>All isolation were successful</p> | ||
+ | <br/> | ||
+ | <p>Task 2:</p> | ||
+ | <ul><li>Isolate the crobox sequence from digested construct</li></ul> | ||
+ | <p>Methods:</p> | ||
+ | <ul><li>After the digestion reaction mixture was loaded on the gel and electrophoretically separated</li></ul> | ||
+ | <p>Results:</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2009/3/3f/Crobox_digest_12_08_09.png" widht="60%" height="60%"> | ||
+ | <font face="Times New Roman" size="3"><p><div style="text-align: center;">Elucidation of the digestion</div></p></font> | ||
+ | <p><b>Comment:</b></p> | ||
+ | <p>There was no sign of digested crobox sequence. Probably the concentration of the crobox DNA was low. To prepare digested sample it is necessary to use more concentrated solution of the crobox. </p><br/> | ||
+ | <p>Task 3:</p> | ||
+ | <ul> | ||
+ | <li>Transformation of chemocompetent E. coli strain DH5α</li> | ||
+ | </ul> | ||
+ | <p><b>Comment:</p></b> | ||
+ | <p>Due to obtain more amount previously cloned constructs the bacteria were transformed with followed constructs: | ||
+ | <ul> | ||
+ | <li>p53 on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li> | ||
+ | <li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li> | ||
+ | <li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li> | ||
+ | <li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_E0032"><span style="color: black">BBa_E0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li></ul><br/> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li>Detailed protocol of transformation is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>.</li> | ||
+ | </ul> | ||
+ | <p>Task 4:</p> | ||
+ | <ul> | ||
+ | <li>Restriction digest of biobricks</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p><strong>Comment:</strong></p> | ||
+ | <p>Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of previously prepared constructs or biobricks were digested:</p> | ||
+ | <p> <a href="http://partsregistry.org/Part:BBa_R0080"><span style="color: black">BBa_R0080</a></span></p> | ||
+ | <p> <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span></p> | ||
+ | <p> cro CDS on pKSII vector </p> | ||
+ | <br/> | ||
+ | <p>Methods:</p><ul> | ||
+ | <li>Digest of BBa_R0080 using SpeI and PstI</li> | ||
+ | <ul><li>Reaction mixture composition: | ||
+ | <pre> | ||
+ | 20 μl purified plasmid DNA product | ||
+ | 0.5 μl SpeI (Fermentas) | ||
+ | 1 μl PstI (Fermentas) | ||
+ | 5 μl Buffer Tango (Fermentas) | ||
+ | 24 μl MQ water | ||
+ | </li></ul></pre> | ||
+ | <li>Digest of other constructs using PstI and XbaI</li> | ||
+ | <ul><li>Reaction mixture composition: | ||
+ | <pre> | ||
+ | 20 μl purified plasmid DNA product | ||
+ | 1 μl XbaI (Fermentas) | ||
+ | 1 μl PstI (Fermentas) | ||
+ | 5 μl Buffer Tango (Fermentas) | ||
+ | 24 μl MQ water</pre> | ||
+ | </li></ul> | ||
+ | <li>All reaction were performed ~8 hours and both of them were subsequently inactivated via heating in 80°C for 20 minutes</li> | ||
+ | <li>The reaction mixtures were loaded on the gel and DNA was electrophoretically separated. In the next step fragments of gel which contain appropriate DNA sequences were cut out and frozen</li><ul> | ||
</html> | </html> | ||
- | |||
Latest revision as of 09:27, 6 September 2009
Assembly of endosomal detection operon
Marcin
Task 1:
- Digest of isolate plasmids to verify the success of isolation
Methods:
- Samples containing plasmids digested in 11.08.2009 were thawed and loaded into the agarose gel
Comment:
Results:
verification of the restriction patterns
Comment
All isolation were successful
Task 2:
- Isolate the crobox sequence from digested construct
Methods:
- After the digestion reaction mixture was loaded on the gel and electrophoretically separated
Results:
Elucidation of the digestion
Comment:
There was no sign of digested crobox sequence. Probably the concentration of the crobox DNA was low. To prepare digested sample it is necessary to use more concentrated solution of the crobox.
Task 3:
- Transformation of chemocompetent E. coli strain DH5α
Comment:
Due to obtain more amount previously cloned constructs the bacteria were transformed with followed constructs:
- p53 on pSB1A3 plasmid
- BBa_B0032+BBa_C0040 on pSB1A3 plasmid
- BBa_B0032+BBa_C0051 on pSB1A3 plasmid
- BBa_B0032+BBa_E0032 on pSB1A3 plasmid
Methods:
- Detailed protocol of transformation is described here.
Task 4:
- Restriction digest of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of previously prepared constructs or biobricks were digested:
cro CDS on pKSII vector
Methods:
- Digest of BBa_R0080 using SpeI and PstI
- Reaction mixture composition:
20 μl purified plasmid DNA product 0.5 μl SpeI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 24 μl MQ water
- Digest of other constructs using PstI and XbaI
- Reaction mixture composition:
20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 24 μl MQ water
- All reaction were performed ~8 hours and both of them were subsequently inactivated via heating in 80°C for 20 minutes
- The reaction mixtures were loaded on the gel and DNA was electrophoretically separated. In the next step fragments of gel which contain appropriate DNA sequences were cut out and frozen
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