Team:Warsaw/Calendar-Main/21 July 2009

From 2009.igem.org

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<h3>Transformation of E. coli with pKS/hly plasmid</h3>
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<h3>Cloning of hly gene into pKSII+ vector</h3>
<h4>Kama</h4>
<h4>Kama</h4>
<ul>
<ul>
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<li>After the heat shock 800&mu;l of SOB medium was added</li>
<li>After the heat shock 800&mu;l of SOB medium was added</li>
<li>Mixture with bacteria was incubated for 1 hour in 37&deg;C</li>
<li>Mixture with bacteria was incubated for 1 hour in 37&deg;C</li>
-
<li>100&mu;l of mixture was plated on medium containing ampicillin, X-Gal and IPTG</li>
+
<li>100&mu;l of mixture was plated on medium containing ampicillin, X-Gal and IPTG (diluted 10* and without dilution)</li>
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<li>The rest of mixture was rotated for 1 minute, pelet was  </li>
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</ul>
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* Gel
* Gel
* DNA samples were cut out and frozen in the seperate eppendorfs (still within the agarose gel).
* DNA samples were cut out and frozen in the seperate eppendorfs (still within the agarose gel).
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[image:https://static.igem.org/mediawiki/2009/4/4b/Lonowanie.jpg]
Results:
Results:
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Comments:  
Comments:  
*There is a vector with PcI clearly visible in the first two samples. The PcI is too short to be visible on its own. The insert, that is RBS.3+LacI is visible as the 1200 band in the last three samples. The vector from two first and the insert from two last samples were cut out of the gel.
*There is a vector with PcI clearly visible in the first two samples. The PcI is too short to be visible on its own. The insert, that is RBS.3+LacI is visible as the 1200 band in the last three samples. The vector from two first and the insert from two last samples were cut out of the gel.
 +
<html>
 +
 +
<h3>Cloning of the mgtc promoter into the pKSII+ plasmid</h3>
 +
<h4>Kamil</h4>
 +
<br />
 +
<p>Tasks:</p>
 +
<ul>
 +
<li>Ligation verification</li>
 +
<li>Bacteria transformation</li>
 +
</ul>
 +
<br />
 +
<p>Methods:</p>
 +
<ul>
 +
<li>Correctness of the ligation was verified with gel electrophoresis on 1% agarose gel.</li>
 +
<li><p>A 200&mu;l batch of chemocompetent bacteria was transformed with 10&mu;l of ligation mix and incubated overnight on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG. (see protocols section for details on transformation). </li>
 +
</ul>
 +
<br />
 +
</html>
 +
 +
<html>
 +
<h3><div style="text-align: center;">Cloning of p53 coding sequence</div></h3>
 +
<h4>Marcin</h4>
 +
<br/>
 +
<p>Task 1:</p>
 +
<ul>
 +
<li>Prepare PCR reaction to amplified p53 coding sequence.</li>
 +
</ul>
 +
<p>Methods:</p>
 +
<ul>
 +
<li>DNA template dilution:</li>
 +
</ul>
 +
<p>1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water</p>
 +
<ul>
 +
<li>PCR mixture composition:</li>
 +
<p>Composition of the mixtures was not changed. Detailed desciption is <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/20_July_2009">here</a> (in the section "Cloning of 53 coding sequence").</p>
 +
</ul>
 +
<ul>
 +
<li>Program:</li></ul>
 +
<br/>
 +
<p>p53 (detailed destription is <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009">here</a>)</p>
 +
<br/>
 +
<p>Results:</p>
 +
<p>No product obtained</p>
 +
<p><b>Comment:</b></p>
 +
<p>It is most likely that Pfu polimerase has been degradated. I decide to prepare this PCR reaction using the Fusion polimerase (NEB) which was kindly provided by dr. Dziembowski.</p><br/>
 +
<ul>
 +
<li>PCR mixture composition:</li></ul>
 +
<ol>
 +
<li>proper mixture 1:
 +
<pre>0.25 &mu;l primer 1 (50 nM; Oligo.pl)
 +
0.25 &mu;l primer 2 (50 nM; Oligo.pl)
 +
1.5 &mu;l dNTPs (20 &mu;M ;Fermentas)
 +
0.25 &mu;l Phusion polymerase (NEB)
 +
5 &mu;l Phusion Buffer (NEB)
 +
2.5 &mu;l MgSO<sub>4</sub> (20 &mu;M; Fermentas)
 +
1 &mu;l DNA template
 +
14.5 &mu;l MQ water</pre></li>
 +
<li>proprer mixture 2:
 +
<pre>0.25 &mu;l primer 1 (50 nM; Oligo.pl)
 +
0.25 &mu;l primer 2 (50 nM; Oligo.pl)
 +
1.5 &mu;l dNTPs (20 &mu;M ;Fermentas)
 +
0.25 &mu;l Phusion polymerase (NEB)
 +
5 &mu;l Phusion Buffer (NEB)
 +
2.5 &mu;l MgSO<sub>4</sub> (20 &mu;M; Fermentas)
 +
2 &mu;l DNA template
 +
13.5 &mu;l MQ water</pre></li>
 +
<li>Negative control: the same as proper mixture 1, the only distinction is lack of the DNA template.</li>
 +
</ol></ul>
 +
<ul>
 +
<li>Program:</li></ul>
 +
<p>p53 (detailed destription is <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009">here</a>)</p>
 +
<p>Results:</p>
 +
<br/><center>
 +
<img src="https://static.igem.org/mediawiki/2009/7/76/P53_PCR_21_07_09.png"></center>
 +
<div style="text-align: center;">PCR product from mixture 2.<br/>Lack of the product from mixture 1<br/>due to high concentration of the template.</div><br/>
 +
<p>Task 2:</p>
 +
<ul>
 +
<li>Clean-up the PCR product and subsequent digest</li></ul>
 +
<p>Methods:</p>
 +
<ul>
 +
<li>DNA was purified using the A&A clean-up kit. Detailed procedure is described <a href="http://www.aabiot.com/products/dna_purification/dna_fragments/clean_up/protocol_clean_up.pdf">here</a></li>
 +
<li>Digest for subsequent cloning using XbaI</li>
 +
<ul><li>Reaction mixture composition:
 +
<pre>10 &mu;l purified PCR product
 +
1 &mu;l XbaI (Fermentas)
 +
5 &mu;l Buffer Tango (Fermentas)
 +
34.5 &mu;l MQ water</pre>
 +
</li></ul>
 +
</ul>
 +
<p>Program:</p>
 +
<p>digest:</p>
 +
<pre>
 +
1. 37&deg;C - 3 hours
 +
2. 80&deg;C - 15 minutes
 +
3. 4&deg;C - hold
 +
</pre>
 +
<ul>
 +
<li>Purification of digested products via gel-out</li>
 +
</ul>
 +
<br/>
 +
<p>Procedure:<p/>
 +
<ul>
 +
<li>Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described <a href="http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf">here</a></li>
 +
 +
</html>
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{{WarNotebookEnd}}
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Latest revision as of 15:03, 1 October 2009


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Cloning of hly gene into pKSII+ vector

Kama

  • chemocompetent E. coli dH5α were incubated on ice for 15 minutes
  • 15μl of ligation mixture was added (and 15μl of control ligation, without insert)
  • bacteria were incubated with DNA on ice for 30 minutes
  • heat shock was conducted (1 minute 42°C)
  • bacteria were incubated on ice for 3 minutes
  • After the heat shock 800μl of SOB medium was added
  • Mixture with bacteria was incubated for 1 hour in 37°C
  • 100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG (diluted 10* and without dilution)

Construction of K177012 operon1_part2

Ania

Tasks:

[image:Lonowanie.jpg] Results:

[image:Ania21.07.gif]

Comments:

  • There is a vector with PcI clearly visible in the first two samples. The PcI is too short to be visible on its own. The insert, that is RBS.3+LacI is visible as the 1200 band in the last three samples. The vector from two first and the insert from two last samples were cut out of the gel.

Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil


Tasks:

  • Ligation verification
  • Bacteria transformation

Methods:

  • Correctness of the ligation was verified with gel electrophoresis on 1% agarose gel.

  • A 200μl batch of chemocompetent bacteria was transformed with 10μl of ligation mix and incubated overnight on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG. (see protocols section for details on transformation).


Cloning of p53 coding sequence

Marcin


Task 1:

  • Prepare PCR reaction to amplified p53 coding sequence.

Methods:

  • DNA template dilution:

1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water

  • PCR mixture composition:

    • Composition of the mixtures was not changed. Detailed desciption is here (in the section "Cloning of 53 coding sequence").

  • Program:

p53 (detailed destription is here)


Results:

No product obtained

Comment:

It is most likely that Pfu polimerase has been degradated. I decide to prepare this PCR reaction using the Fusion polimerase (NEB) which was kindly provided by dr. Dziembowski.


  • PCR mixture composition:
  1. proper mixture 1: 
    0.25 μl primer 1 (50 nM; Oligo.pl) 
0.25 μl primer 2 (50 nM; Oligo.pl) 
1.5 μl dNTPs (20 μM ;Fermentas) 
0.25 μl Phusion polymerase (NEB) 
5 μl Phusion Buffer (NEB) 
2.5 μl MgSO4 (20 μM; Fermentas) 
1 μl DNA template 
14.5 μl MQ water
  2. proprer mixture 2: 
    0.25 μl primer 1 (50 nM; Oligo.pl) 
0.25 μl primer 2 (50 nM; Oligo.pl) 
1.5 μl dNTPs (20 μM ;Fermentas)
0.25 μl Phusion polymerase (NEB)
5 μl Phusion Buffer (NEB)
2.5 μl MgSO4 (20 μM; Fermentas)
2 μl DNA template 
13.5 μl MQ water
  3. Negative control: the same as proper mixture 1, the only distinction is lack of the DNA template.
  • Program:

p53 (detailed destription is here)

Results:


PCR product from mixture 2.
Lack of the product from mixture 1
due to high concentration of the template.

Task 2:

  • Clean-up the PCR product and subsequent digest

Methods:

  • DNA was purified using the A&A clean-up kit. Detailed procedure is described here
  • Digest for subsequent cloning using XbaI
    • Reaction mixture composition: 
      10 μl purified PCR product 
1 μl XbaI (Fermentas) 
5 μl Buffer Tango (Fermentas) 
34.5 μl MQ water

Program:

digest:

1. 37°C - 3 hours
2. 80°C - 15 minutes
3. 4°C - hold
  • Purification of digested products via gel-out

Procedure:

Retrieved from "http://2009.igem.org/Team:Warsaw/Calendar-Main/21_July_2009"