Team:Warsaw/Calendar-Main/3 September 2009
From 2009.igem.org
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pSB6A1 | pSB6A1 | ||
+ | |||
+ | |||
+ | <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | ||
+ | <h4>Marcin</h4> | ||
+ | |||
+ | Task 1: | ||
+ | * Isolate the plasmids containing following construct: | ||
+ | * [http://partsregistry.org/Part:BBa_K177035<span style="color: black">BBa_K177035</span>] on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] plasmid | ||
+ | Methods: | ||
+ | Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here] | ||
+ | |||
+ | Task 2: | ||
+ | * Digestion of plasmids previously described constructs | ||
+ | Methods: | ||
+ | * Reaction mixture composition: | ||
+ | <pre> | ||
+ | 20 μl purified plasmid DNA product | ||
+ | 1 μl XbaI (Fermentas) | ||
+ | 1 μl PstI (Fermentas) | ||
+ | 2 μl Buffer Tango (Fermentas) | ||
+ | 15 μl MQ water | ||
+ | </pre> | ||
+ | * Digest was performed about six hours and subsequently thermal inactivated | ||
+ | |||
+ | Task 3: | ||
+ | * Gel-outs of construct described in Task 2 | ||
+ | Methods: | ||
+ | * All gel-outs were performed using the EurX gel-out kit according to the manual | ||
+ | |||
+ | Task 4: | ||
+ | |||
+ | * Ligation of following constructs: | ||
+ | ** [http://partsregistry.org/Part:BBa_K177037<span style="color: black">BBa_K177037</span>] on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] plasmid | ||
+ | ** cro CDS on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] | ||
+ | ** Bax CDS on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] | ||
+ | Methods: | ||
+ | * Reaction mixture composition: | ||
+ | <pre> | ||
+ | 10 μl digested insert | ||
+ | 8 μl digested vector | ||
+ | 2 μl Tango buffer(Fermentas) | ||
+ | 3 μl dNTPs mixture (EurX, concentration 5 mM) | ||
+ | 1 μl ligase T4 (Fermentas) | ||
+ | </pre> | ||
+ | * Reaction were carried out 12 hours and subsequently thermally inactivated. | ||
+ | |||
+ | <html> | ||
+ | <h3><div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div></h3> | ||
+ | <h4>Kamil</h4> | ||
+ | <br /> | ||
+ | <p>Tasks:</p> | ||
+ | <ul> | ||
+ | <li>Plasmid isolation</li> | ||
+ | <li>Control digest</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li>Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).</li> | ||
+ | <li>The digest mix was prepared as follows: | ||
+ | <p><pre>20μl isolated plasmid<br/>3μl Buffer O (Fermentas)<br/>1μl PstI enzyme<br/>1μl EcoRI enzyme<br/>5μl H<sub>2</sub>O</pre></p></li> | ||
+ | <li>The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.</li> | ||
+ | <li>The results were visualised on 1% agarose gel</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Results:</p> | ||
+ | <ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2009/6/65/2009.09.03.jpg"> | ||
+ | <li>First four bands (besides the the size ruler) show empty plasmids.</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p>Conclusions:</p> | ||
+ | <ul> | ||
+ | <li>Respawn and try again in... 3 days.</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | |||
+ | |||
+ | <h3><div style="text-align: center;">Cloning of the cro-box into the pSB1A3 plasmid</div></h3> | ||
+ | <h4>Kamil</h4> | ||
+ | <br /> | ||
+ | <p>Tasks:</p> | ||
+ | <ul> | ||
+ | <li>Plasmid isolation</li> | ||
+ | <li>Control digest</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li>Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).</li> | ||
+ | <li>The digest mix was prepared as follows: | ||
+ | <p><pre>20μl isolated plasmid<br/>3μl Buffer O (Fermentas)<br/>1μl PstI enzyme<br/>1μl EcoRI enzyme<br/>5μl H<sub>2</sub>O</pre></p></li> | ||
+ | <li>The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.</li> | ||
+ | <li>The results were visualised on 1% agarose gel</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Results:</p> | ||
+ | <ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2009/6/65/2009.09.03.jpg"> | ||
+ | <li>Final five bands show empty plasmids.</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p>Conclusions:</p> | ||
+ | <ul> | ||
+ | <li>Respawn and try again in... 3 days.</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <h3><div style="text-align: center;">Cotransformation to obtain cells with following plasmid combinations [<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177038">K177038</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177038">K177038</a>] | ||
+ | <h4>Ania</h4> | ||
+ | <br/> | ||
+ | <p>Tasks:</p> | ||
+ | <ul> | ||
+ | <li> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | </html> | ||
Latest revision as of 22:16, 20 September 2009
Contents |
Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek
Tasks:
- Digestion of PCR products, separetly with MboI and CaiI restriction endonucleases
- Electrophoretical segregation of digested samples in 1,5% agarose gel
- Preparation of another PCR reaction with L.monocytogenes genome DNA
Results:
- There were no visible products of digestion, even after long treatment with ethidium bromide. Propably the Clean-Up kit isn't working well. I'll try to acquire IntA gene with another PCR.
Preparation of glycerol stocks
Preparation of glycerol stocks
Monika
- Preparation of glycerol stocks following the prodcedure described here
pAraC + GFP
plac + RBS + cI + ter
plac + RBS + cI + RBS + GFP + ter
J07037
plac
pSB6A1
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmids containing following construct:
- BBa_K177035 on pSB1A3 plasmid
Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Task 2:
- Digestion of plasmids previously described constructs
Methods:
- Reaction mixture composition:
20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
- Digest was performed about six hours and subsequently thermal inactivated
Task 3:
- Gel-outs of construct described in Task 2
Methods:
- All gel-outs were performed using the EurX gel-out kit according to the manual
Task 4:
- Ligation of following constructs:
- BBa_K177037 on pSB1A3 plasmid
- cro CDS on pSB1A3
- Bax CDS on pSB1A3
Methods:
- Reaction mixture composition:
10 μl digested insert 8 μl digested vector 2 μl Tango buffer(Fermentas) 3 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas)
- Reaction were carried out 12 hours and subsequently thermally inactivated.
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Plasmid isolation
- Control digest
Methods:
- Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
- The digest mix was prepared as follows:
20μl isolated plasmid
3μl Buffer O (Fermentas)
1μl PstI enzyme
1μl EcoRI enzyme
5μl H2O - The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
- The results were visualised on 1% agarose gel
Results:
- First four bands (besides the the size ruler) show empty plasmids.
Conclusions:
- Respawn and try again in... 3 days.
Cloning of the cro-box into the pSB1A3 plasmid
Kamil
Tasks:
- Plasmid isolation
- Control digest
Methods:
- Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
- The digest mix was prepared as follows:
20μl isolated plasmid
3μl Buffer O (Fermentas)
1μl PstI enzyme
1μl EcoRI enzyme
5μl H2O - The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
- The results were visualised on 1% agarose gel
Results:
- Final five bands show empty plasmids.
Conclusions:
- Respawn and try again in... 3 days.
Cotransformation to obtain cells with following plasmid combinations [K177012,K177011][K177012,K177038][K177033,K177011][K177033,K177038]
Ania
Tasks:
-
April
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June
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August
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September
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Ania
Tasks:
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