Team:Warsaw/Calendar-Main/11 August 2009

From 2009.igem.org

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<p>Preparation of bacterial cultures</p>
<p>Preparation of bacterial cultures</p>
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<ul>
<li>Prepare LB medium with ampicillin</li>
<li>Prepare LB medium with ampicillin</li>
<li>Add 3.5 ml of the medium to the probes</li>
<li>Add 3.5 ml of the medium to the probes</li>
<li>Add one bacterial colony to each probe</li>
<li>Add one bacterial colony to each probe</li>
<li>Breed the bacteria about 7 hours</li>
<li>Breed the bacteria about 7 hours</li>
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<p>Task 2:</p>
<p>Task 2:</p>
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<p>Methods:</p><ul>
<p>Methods:</p><ul>
<li>Digest of isolate plasmids using EcoRI and PstI</li>
<li>Digest of isolate plasmids using EcoRI and PstI</li>
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<ul><li>Reaction mixture composition: 1 &mu;l purified plasmid DNA product, 0.5 &mu;l XbaI (Fermentas),0.5 &mu;l PstI (Fermentas), 2 &mu;l Buffer Orange (Fermentas), 16 &mu;l MQ water</li></ul>
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<ul><li>Reaction mixture composition:  
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<pre>1 &mu;l purified plasmid DNA product
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0.5 &mu;l XbaI (Fermentas)
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0.5 &mu;l PstI (Fermentas)
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2 &mu;l Buffer Orange (Fermentas)
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16 &mu;l MQ water</pre>
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</li></ul>
<li>The reaction was performed two hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li>
<li>The reaction was performed two hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li>
<li>After the inactivation samples were frozen in -20&deg;c</li></ul><br/>
<li>After the inactivation samples were frozen in -20&deg;c</li></ul><br/>
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<li>Reaction mixture composition: 20 &mu;l purified plasmid solution , 0.75 &mu;l XbaI (Fermentas),0.75 &mu;l PstI (Fermentas), 5 &mu;l Buffer Orange (Fermentas), 23.5 &mu;l MQ water</li>
<li>Reaction mixture composition: 20 &mu;l purified plasmid solution , 0.75 &mu;l XbaI (Fermentas),0.75 &mu;l PstI (Fermentas), 5 &mu;l Buffer Orange (Fermentas), 23.5 &mu;l MQ water</li>
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<li>The reaction was performed twenty hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li>
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<li>The reaction was performed twenty hours and was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li>
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Revision as of 19:23, 13 August 2009


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Assembly of endosomal detection operon

Marcin


Task 1:

  • Prepare the bacterial cultures for isolation of plasmid containing BBa_R0080 and BBa_E0022

Preparation of bacterial cultures

  • Prepare LB medium with ampicillin
  • Add 3.5 ml of the medium to the probes
  • Add one bacterial colony to each probe
  • Breed the bacteria about 7 hours

Task 2:

  • Isolate the plasmid containing BBa_R0080 and BBa_E0022

    • Methods:

  • Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 3:

  • Digest of isolate plasmids verify the success of isolation

Methods:

  • Digest of isolate plasmids using EcoRI and PstI
    • Reaction mixture composition: 
      1 μl purified plasmid DNA product
0.5 μl XbaI (Fermentas)
0.5 μl PstI (Fermentas)
2 μl Buffer Orange (Fermentas)
16 μl MQ water
  • The reaction was performed two hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
  • After the inactivation samples were frozen in -20°c

Task 4:

  • Transformation of chemocompetent E. coli strain DH5α

Methods:

  • Ligation prepared in 08.08.09 was stopped via thermal inactivation in 80°C for 20 minutes
  • Detailed protocol of transformation is described here.

Task 5:

  • Isolate the crobox sequence from digested construct

Methods:

  • After the digestion reaction mixture was loaded on the gel and electrophoretically separated

Comment:

There was no sign of digested crobox sequence. Probably the concentration of the DNA was low or agarose used to prepare the gel was partially degraded. The digestion should be prepare another time.


Task 6:

  • Digest pKS vector to isolate crobox sequence

Methods:

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