Team:Warsaw/Calendar-Main/7 September 2009
From 2009.igem.org
(Difference between revisions)
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</pre> | </pre> | ||
* Ligation was carry out about 15 hours | * Ligation was carry out about 15 hours | ||
+ | <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>Ligation</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li>The ligation mix was prepared as follows: | ||
+ | <p><div align=center><pre>20μl purified plasmid backbone<br/>10μl purified PCR product<br/>4μl Ligase buffer (Fermentas)<br/>1μl T4 Ligase (Fermentas)<br/>5μl H<sub>2</sub>O</pre></li> | ||
+ | <li>The ligation was carried out in 18°C for 3h and then inactivated in 80°C for 15 min.</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>Ligation to the plasmid</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li>The ligation mix was prepared as follows: | ||
+ | <p><div align=center><pre>10μl purified plasmid backbone<br/>10μl purified PCR product<br/>3μl Ligase buffer (Fermentas)<br/>1μl T4 Ligase (Fermentas)<br/>6μl H<sub>2</sub>O</pre></li> | ||
+ | <li>The ligation was carried out in 18°C for 3h and then inactivated in 80°C for 15 min.</li> | ||
+ | <br /> | ||
+ | </html> | ||
<!-- do not remove this! --> | <!-- do not remove this! --> | ||
{{WarNotebookEnd}} | {{WarNotebookEnd}} |
Revision as of 21:33, 8 September 2009
Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek
Tasks:
- Electrophoretical segregation of PCR products from previous day in 0,8% agarosis gel (sample containing 10 microL was used).
- Purifing DNA from agarose block with A&A Gel-Out kit.
Results:
- Finally I have the correct PCR product (at least I hope it's that one).
Assembly of endosomal detection operon
Marcin
Task 1:
- Digest pKSII cloning vector using SmaI:
Methods:
- Reaction mixture composition:
15 μl purified plasmid DNA product 0.5 μl SmaI (Fermentas) 2 μl Buffer Tango (Fermentas) 12.5 μl MQ water
- Digestion was performed 6 hours and in next step enzyme was inactivated in 65 °C for 20 minutes
Task 2:
- Phosphorylation of previously purified sequence of mitochondrial signal peptide from Cox1 :
Methods:
- Reaction mixture composition:
15 μl purified plasmid DNA product 2 μl PNK Buffer (NEB) 0.5 μl PNK (NEB) 2.5 μl dNTPs mixture (EurX, concentration 5 mM)
- Reaction was performed 45 minutes and subsequently inactivate via heating (65 °C)
Task 3: Ligate of mitochondrial signal peptide to pKSII vector
- Methods:
- Ligation mixtures composition:
10 μl phosphorylated insert 8 μl digested vector 2.5 μl Tango buffer(Fermentas) 2.5 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas)
- Ligation was carry out about 15 hours
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Ligation
Methods:
- The ligation mix was prepared as follows:
20μl purified plasmid backbone
10μl purified PCR product
4μl Ligase buffer (Fermentas)
1μl T4 Ligase (Fermentas)
5μl H2O- The ligation was carried out in 18°C for 3h and then inactivated in 80°C for 15 min.
Cloning of the cro-box into the pSB1A3 plasmidKamil
Tasks:
- Ligation to the plasmid
Methods:
- The ligation mix was prepared as follows:
10μl purified plasmid backbone
10μl purified PCR product
3μl Ligase buffer (Fermentas)
1μl T4 Ligase (Fermentas)
6μl H2O- The ligation was carried out in 18°C for 3h and then inactivated in 80°C for 15 min.
April M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 May M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 June M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 July M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 August M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 September M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 October M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31