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Team:Warsaw/Calendar-Main/15 July 2009
From 2009.igem.org
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* 4 positive [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>], and 16 [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>] were found. | * 4 positive [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>], and 16 [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>] were found. | ||
| - | * Each clone was transferred to new plate with LB, agar-agar and IPTG or 0. | + | * Each clone was transferred to new plate with LB, agar-agar and IPTG or 0.2 % arabinose. Liquid cultures were also established. |
<br> | <br> | ||
| Line 50: | Line 50: | ||
<p>Conclusions:</p> | <p>Conclusions:</p> | ||
<ul> | <ul> | ||
| - | <li>The transformation needs to be redone, this time using more of the ligation mix. | + | <li>The transformation needs to be redone, this time using more of the ligation mix.</li> |
| + | </ul> | ||
</var> | </var> | ||
| Line 57: | Line 58: | ||
</html> | </html> | ||
| + | <html> | ||
| - | + | <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | |
| - | + | <h4>Marcin</h4> | |
| - | + | <br/> | |
| - | + | <p>Task 1:</p> | |
| - | Task 1: | + | <ul> |
| - | + | <li>Isolate plasmids containing the biobricks</li> | |
| - | + | </ul> | |
| - | + | <br/> | |
| - | Methods: | + | <p>Methods:</p> |
| - | + | <ul> | |
| - | + | <li>Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described | |
| - | + | <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf">here</a>.</li> | |
| - | + | <li>After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel</li> | |
| - | voltage - 70V | + | <li>Electrophoresis condition:</li> |
| - | + | </ul> | |
| - | time - 30 min | + | <p>voltage - 70V</p> |
| - | + | <p>time - 30 min</p> | |
| - | + | <ul> | |
| - | + | <li>Next the gel was photographed:</li> | |
| - | + | </ul> | |
| - | Legend:< | + | <br/> |
| - | 1-3 - | + | <center> |
| - | + | <img src="https://static.igem.org/mediawiki/2009/4/41/Biobricks_izolacja_15_07_09.png" width="60%" height="60%"> | |
| - | 4-6 - | + | </center> |
| - | + | <div style="text-align: center";>Samples of plasmids after isolation</div> | |
| - | 7-9 - | + | <br> |
| - | + | <p>Legend:</p> | |
| - | 10-12 - | + | <p>1-3 - <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span></p> |
| - | + | <p>4-6 - <a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_c0040</a></span></p> | |
| - | + | <p>7-9 - <a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span></p> | |
| - | + | <p>10-12 - <a href="http://partsregistry.org/Part:BBa_E0032"><span style="color: black">BBa_E0032</a></span></p> | |
| - | Isolation of plasmids was successful. | + | <p><strong>Comment:</strong></p> |
| - | + | <p>Isolation of plasmids was successful.</p> | |
| - | + | </br> | |
| - | + | <h3><div style="text-align: center;">Cloning of p53 coding sequence</div></h3> | |
| - | + | <h4>Marcin</h4> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| + | <p>Task:</p> | ||
| + | <ul> | ||
| + | <li>Cloning p53 coding sequence to pKS plasmid</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <li>Ligation mixture composition: | ||
| + | <pre>14 μl digested p53 | ||
| + | 1.5 μl digested pKS | ||
| + | 5 μl ligation buffer (Invitrogen) | ||
| + | 1 μl ligase T4</pre></li> | ||
| + | <li>Duration of ligation was about 20 hours; reaction was conducted in 16 °C (approximately).</li> | ||
| + | </ul> | ||
| + | </html> | ||
| + | <html> | ||
| + | <h3>Insertion of the pho gene into the pKSII+ plasmid</h3> | ||
| + | <h4>Kama</h4> | ||
| + | <ul> | ||
| + | <li>Isolation of plasmid containing an insert was performed with the <a href=http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf>A&A plasmid mini kit</a></li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <img src="https://static.igem.org/mediawiki/2009/8/87/2009_07_16_pKS_pho_opisany.JPG"> | ||
| + | <br/> | ||
| + | <p>Notes</p> | ||
| + | <ul> | ||
| + | <li>All samples contain empty plasmid</li> | ||
| + | </ul> | ||
| + | </html> | ||
| + | <h3> <div style="text-align: center;">Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2 </div></h3> | ||
| + | <h4>Ania</h4> | ||
| + | Tasks: | ||
| + | |||
| + | * Electrophoresis, gel extraction and ligation of: | ||
| + | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert) | ||
| + | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector) | ||
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Latest revision as of 22:32, 20 September 2009
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Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])
Franek
Tasks:
- Selecting clones with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Methods:
- Tranformants with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] clones were recognised due to the fluorescence under UV light.
- 4 positive [http://partsregistry.org/Part:BBa_K177024 BBa_K177024], and 16 [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] were found.
- Each clone was transferred to new plate with LB, agar-agar and IPTG or 0.2 % arabinose. Liquid cultures were also established.
Results:
- [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] were successfully created!
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Bacteria transformation
Methods:
- A 200μl batch of chemocompetent bacteria was transformed with 15μl of ligation mix and incubated overnight on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.
Results:
The transformation yielded only a couple of blue colonies.
Conclusions:
- The transformation needs to be redone, this time using more of the ligation mix.
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate plasmids containing the biobricks
Methods:
- Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here.
- After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel
- Electrophoresis condition:
voltage - 70V
time - 30 min
- Next the gel was photographed:
Samples of plasmids after isolation
Legend:
1-3 - BBa_B0032
4-6 - BBa_c0040
7-9 - BBa_C0051
10-12 - BBa_E0032
Comment:
Isolation of plasmids was successful.
Cloning of p53 coding sequence
Marcin
Task:
- Cloning p53 coding sequence to pKS plasmid
Methods:
- Ligation mixture composition:
14 μl digested p53 1.5 μl digested pKS 5 μl ligation buffer (Invitrogen) 1 μl ligase T4
- Duration of ligation was about 20 hours; reaction was conducted in 16 °C (approximately).
Insertion of the pho gene into the pKSII+ plasmid
Kama
- Isolation of plasmid containing an insert was performed with the A&A plasmid mini kit
Notes
- All samples contain empty plasmid
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Electrophoresis, gel extraction and ligation of:
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert)
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)
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