Team:Warsaw/Calendar-Main/14 August 2009

From 2009.igem.org

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<html>
<html>
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<h3>Cloning Bax into pBS plasmid</h3>
+
<h3>Cloning Bax into pSB1A3 plasmid</h3>
<h4>Justyna</h4>
<h4>Justyna</h4>
<p>Task 1:</p>
<p>Task 1:</p>
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<p>Task 2:</p>
<p>Task 2:</p>
<ul>
<ul>
-
<li>Digestion and gel-out of pBS plasmid</li>
+
<li>Digestion and gel-out of pSB1A3 plasmid</li>
</ul>
</ul>
<br/>
<br/>
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<p>Task 3:</p>
<p>Task 3:</p>
<ul>
<ul>
-
<li>Ligation of Bax and pBS</li></ul>
+
<li>Ligation of Bax and pSB1A3</li></ul>
<p>Methods:</p><ul>
<p>Methods:</p><ul>
<li>Ligation mix:</li>
<li>Ligation mix:</li>
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2.5 &mu;l - Tango buffer (Fermentas, 10x)
2.5 &mu;l - Tango buffer (Fermentas, 10x)
2.0 &mu;l - dNTPs (EURx, 5&mu;lM)
2.0 &mu;l - dNTPs (EURx, 5&mu;lM)
-
5.0 &mu;l - digested pSB (~100 ng)
+
5.0 &mu;l - digested pSB1A3 (~100 ng)
14.5 &mu;l - Bax (~ 105 ng)
14.5 &mu;l - Bax (~ 105 ng)
in 25.0 &mu;ll
in 25.0 &mu;ll
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<p>Task 4:</p>
<p>Task 4:</p>
<ul>
<ul>
-
<li>Insertion of Bax-pBS plasmid into bacteria</li></ul>
+
<li>Insertion of Bax-pSB1A3 plasmid into bacteria</li></ul>
<p>Methods:</p><ul>
<p>Methods:</p><ul>
<li>Previously prepared chemicompetent bacteria were used.</li>
<li>Previously prepared chemicompetent bacteria were used.</li>
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<ul><li> Isolate the plasmid prepared in <a href="https://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/13_August_2009">13.08.09</a></ul></li>
<ul><li> Isolate the plasmid prepared in <a href="https://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/13_August_2009">13.08.09</a></ul></li>
<p>Methods:</p><ul>
<p>Methods:</p><ul>
-
<li>Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf" here>here</a></li></ul>
+
<li>Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf">here</a></li></ul>
<br/>
<br/>
<p>Task 2:</p><ul><li>Restriction digest of following sequences from the <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid:
<p>Task 2:</p><ul><li>Restriction digest of following sequences from the <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid:
Line 85: Line 85:
<p>Methods:<p/>
<p>Methods:<p/>
<ul>
<ul>
-
<li>Fragments of agarose gel were carefully cut out and subsequently frozen in -20&deg;C./gel_out/protocol_gel_out.pdf">here</a></li></ul>
+
<li>Fragments of agarose gel were carefully cut out and subsequently frozen in -20&deg;C></li></ul>
 +
<p>Results</p>
 +
<center><img src="https://static.igem.org/mediawiki/2009/9/9a/P53_R0080_E0022_C0040_B0032_digest_14_08_09.png" width="40%" height="40%"></center>
 +
<font face="Times New Roman" size="3"><p><div style="text-align: center;">Verification of the digestions</div></p></font>
<p><b>Comment:</b></p>
<p><b>Comment:</b></p>
<p>Restriction digests of samples containing <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> and  <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span></li> were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest. <p><br/>
<p>Restriction digests of samples containing <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> and  <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span></li> were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest. <p><br/>
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<p>Task 6:</p><ul><li>Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)</ul></li>
<p>Task 6:</p><ul><li>Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)</ul></li>
<p>Methods:<p/><ul>
<p>Methods:<p/><ul>
-
<li>Detailed protocol is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/22_July_2009">here</a>
+
<li>Detailed protocol is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/22_July_2009">here</a></li>

Latest revision as of 15:48, 28 September 2009


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Cloning Bax into pSB1A3 plasmid

Justyna

Task 1:

  • Gel-out Bax PCR product

Methods:

  • Bax PCR products were isolated from agarose gel using A&A Gel-Out kit.


Task 2:

  • Digestion and gel-out of pSB1A3 plasmid

Methods:

  • Digestion of isolated plasmids by XbaI and SpeI
  • Reaction mixture composition: 10.0 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl SpeI (Fermentas), 2 μl Buffer Orange (Fermentas), 7.0 μl MQ water
  • The digestion was performed overnight and it was subsequently inactivated via heating in 80°C for 20 minutes.
  • In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids and isolated using A&A Gel-Out kit

Task 3:

  • Ligation of Bax and pSB1A3

Methods:

  • Ligation mix:
    • 1.0 μl - T4 ligase
2.5 μl - Tango buffer (Fermentas, 10x)
2.0 μl - dNTPs (EURx, 5μlM)
5.0 μl - digested pSB1A3 (~100 ng)
14.5 μl - Bax (~ 105 ng)
in 25.0 μll
  • Ligation time - overnight


Task 4:

  • Insertion of Bax-pSB1A3 plasmid into bacteria

Methods:

  • Previously prepared chemicompetent bacteria were used.





Assembly of endosomal detection operon

Marcin


Task 1:

  • Isolate the plasmid prepared in 13.08.09

Methods:

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

Task 2:

Methods:

  • Reaction mixture composition:
    •  
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas) or 0,5 μl SpeI (Fermentas) in the case of R0080
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
24 μl MQ water

Task 3:

  • Gel-out of digest sequences described in Task 1

Methods:

  • Fragments of agarose gel were carefully cut out and subsequently frozen in -20°C>

Results

Verification of the digestions

Comment:

Restriction digests of samples containing BBa_E0022 and BBa_B0032+BBa_C0040 were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest.


Task 4:

  • Another digest of E0022 and C0040+B0032

Methods:

  • Reaction mixture composition:
    •  
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
24 μl MQ water

Task 5:

  • Transformation of chemocompetent E. coli strain DH5&alpha

Constructs to transform:

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 6:

  • Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)

Methods:

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