Team:Warsaw/Calendar-Main/16 July 2009
From 2009.igem.org
(Difference between revisions)
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<li>Both the dish and the liquid cultures were incubated at 37°C overnight. | <li>Both the dish and the liquid cultures were incubated at 37°C overnight. | ||
</ul> | </ul> | ||
+ | <br /> | ||
+ | <p>Results:</p> | ||
+ | <ul> | ||
+ | <li>All of the transferred colonies turned blue overnight and the purified plasmids yealded no mgtc promoter. | ||
+ | </ul> | ||
+ | <br /> | ||
+ | |||
Line 33: | Line 40: | ||
<ul> | <ul> | ||
<li>Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes</li> | <li>Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes</li> | ||
- | <li>Detailed protocol of | + | <li>Detailed protocol of transformation is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is usage of half volume of ligation mixture prepared 15.07.09</li> |
</ul> | </ul> | ||
Line 46: | Line 53: | ||
<br/> | <br/> | ||
<p><strong>Comment:</strong></p> | <p><strong>Comment:</strong></p> | ||
- | <p>Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested: | + | <p>Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:</p> |
<p> <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span></p> | <p> <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span></p> | ||
<p> <a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_c0040</a></span></p> | <p> <a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_c0040</a></span></p> | ||
Line 54: | Line 61: | ||
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
<li>Digest of BBa_B0032 using SpeI and PstI</li> | <li>Digest of BBa_B0032 using SpeI and PstI</li> | ||
- | <ul><li>Reaction mixture composition: 10 μl purified plasmid DNA product | + | <ul><li>Reaction mixture composition: |
+ | <pre>10 μl purified plasmid DNA product | ||
+ | 0.5 μl SpeI (Fermentas) | ||
+ | 1 μl PstI (Fermentas) | ||
+ | 5 μl Buffer Tango (Fermentas) | ||
+ | 34 μl MQ water</pre></li></ul> | ||
<li>Digest of other biobricks using PstI and XbaI</li> | <li>Digest of other biobricks using PstI and XbaI</li> | ||
- | <ul><li> | + | <ul><li>Reaction mixture composition: |
- | <li>Both reaction were performed overnight (~12 hours) and both of them were subsequently inactivated via heating in | + | <pre>10 μl purified plasmid DNA product |
- | + | 1 μl XbaI (Fermentas) | |
+ | 1 μl PstI (Fermentas) | ||
+ | 5 μl Buffer Tango (Fermentas) | ||
+ | 34 μl MQ water</pre></li></ul> | ||
+ | <li>Both reaction were performed overnight (~12 hours) and both of them were subsequently inactivated via heating in 80°C for 20 minutes</li> | ||
+ | </ul></li> | ||
</html> | </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: | ||
+ | |||
+ | * Set up a liquid culture from transformants containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid. | ||
+ | |||
+ | * Alkaline lysis of bacterial cultures to obtain plasmid. | ||
+ | |||
+ | * Digest of the ligated [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid with EcoRI and PstI to confirm ligation. | ||
+ | |||
+ | |||
+ | |||
+ | <!-- TU EDYTUJE FRANEK, NIE RUSZ! --> | ||
+ | ===<div style="text-align: center;">Testing different <em>E. coli</em> strains regarding [http://partsregistry.org/Part:BBa_R0010 <span style="color: black;">lacI</span>] and [http://partsregistry.org/Part:BBa_R0080 <span style="color: black;">AraC</span>] repressors</div>=== | ||
+ | |||
+ | '''Franek''' | ||
+ | |||
+ | <br> | ||
+ | Tasks: | ||
+ | |||
+ | * Alkaline lysis of bacterial cultures to obtain [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>] and [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>] devices | ||
+ | |||
+ | * Transformation of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>] and [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>] | ||
+ | |||
+ | <br> | ||
+ | Methods: | ||
+ | |||
+ | * 2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>] and [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>] on [http://partsregistry.org/Part:pSB1A2 <span style="color: black;">pSB1A2</span>] plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our [https://2009.igem.org/Wiki/Team:Warsaw/protocols <span style="color: black;">standard procedure</span>]. The pellet from 5 ml of bacteria was used. | ||
+ | |||
+ | * Chemocompetent <em>E. coli</em> cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains (prepered earlier by Kuba) were transformed according to our [https://2009.igem.org/Wiki/Team:Warsaw/protocols <span style="color: black;">standard procedure</span>] with either [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>] or [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>] | ||
+ | |||
+ | <br> | ||
+ | Results: | ||
+ | * Will be determined by cell colonies presence on plates | ||
+ | <!-- TU PISZ CO CHCESZ! --> | ||
<!-- do not remove this! --> | <!-- do not remove this! --> | ||
{{WarNotebookEnd}} | {{WarNotebookEnd}} |
Latest revision as of 22:36, 20 September 2009
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Transformant selection
Methods:
- White colonies were picked up and transferred to a new petri dish.
- Liquid cultures were established along the way for plasmid purification.
- Both the dish and the liquid cultures were incubated at 37°C overnight.
Results:
- All of the transferred colonies turned blue overnight and the purified plasmids yealded no mgtc promoter.
Cloning of p53 coding sequence
Marcin
Task:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of transformation is described here. The only modification is usage of half volume of ligation mixture prepared 15.07.09
Assembly of endosomal detection operon
Marcin
Task:
- Restriction digest of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:
Methods:
- Digest of BBa_B0032 using SpeI and PstI
- Reaction mixture composition:
10 μl purified plasmid DNA product 0.5 μl SpeI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 34 μl MQ water
- Digest of other biobricks using PstI and XbaI
- Reaction mixture composition:
10 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 34 μl MQ water
- Both reaction were performed overnight (~12 hours) and both of them were subsequently inactivated via heating in 80°C for 20 minutes
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Set up a liquid culture from transformants containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid.
- Alkaline lysis of bacterial cultures to obtain plasmid.
- Digest of the ligated [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid with EcoRI and PstI to confirm ligation.
Testing different E. coli strains regarding [http://partsregistry.org/Part:BBa_R0010 lacI] and [http://partsregistry.org/Part:BBa_R0080 AraC] repressors
Franek
Tasks:
- Alkaline lysis of bacterial cultures to obtain [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] devices
- Transformation of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Methods:
- 2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.
- Chemocompetent E. coli cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains (prepered earlier by Kuba) were transformed according to our standard procedure with either [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] or [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Results:
- Will be determined by cell colonies presence on plates
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