Team:Warsaw/Calendar-Main/7 September 2009

From 2009.igem.org

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<p><div align=center><pre>20&mu;l purified plasmid backbone<br/>10&mu;l purified PCR product<br/>4&mu;l Ligase buffer (Fermentas)<br/>1&mu;l T4 Ligase (Fermentas)<br/>5&mu;l H<sub>2</sub>O</pre></li>
<p><div align=center><pre>20&mu;l purified plasmid backbone<br/>10&mu;l purified PCR product<br/>4&mu;l Ligase buffer (Fermentas)<br/>1&mu;l T4 Ligase (Fermentas)<br/>5&mu;l H<sub>2</sub>O</pre></li>
<li>The ligation was carried out in 18&deg;C for 3h and then inactivated in 80&deg;C for 15 min.</li>
<li>The ligation was carried out in 18&deg;C for 3h and then inactivated in 80&deg;C for 15 min.</li>
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<li>A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin</li>
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<li>A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin.</li>
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<p><div align=center><pre>10&mu;l purified plasmid backbone<br/>10&mu;l purified PCR product<br/>3&mu;l Ligase buffer (Fermentas)<br/>1&mu;l T4 Ligase (Fermentas)<br/>6&mu;l H<sub>2</sub>O</pre></li>
<p><div align=center><pre>10&mu;l purified plasmid backbone<br/>10&mu;l purified PCR product<br/>3&mu;l Ligase buffer (Fermentas)<br/>1&mu;l T4 Ligase (Fermentas)<br/>6&mu;l H<sub>2</sub>O</pre></li>
<li>The ligation was carried out in 18&deg;C for 3h and then inactivated in 80&deg;C for 15 min.</li>
<li>The ligation was carried out in 18&deg;C for 3h and then inactivated in 80&deg;C for 15 min.</li>
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<li>A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin</li>
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<li>A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin.</li>
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Revision as of 21:38, 8 September 2009


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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
  • Bacteria transformation

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.
  • A fresh batch of chemocompetent bacteria was transformed with the ligation mix and incubated on agarose plates containing double dose of ampicilin.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Ligation to the plasmid
  • Bacteria transformation

Methods:

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