Team:Warsaw/Calendar-Main/13 July 2009

From 2009.igem.org

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===Cloning the p53 coding sequence===
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<html>
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<h3>Cloning the p53 coding sequence</h3>
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'''Marcin'''
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<h4>Marcin</h4>
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<br/>
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Task 1:
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<p>Task 1:</p>
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<ul>
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*Prepare PCR reaction to amplified p53 coding sequence.
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<li>Prepare PCR reaction to amplified p53 coding sequence.</li>
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Methods:
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</ul>
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* DNA matrix dilution:
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<p>Methods:</p>
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1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water
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<ul>
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* PCR mixture composition:
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<li>DNA template dilution:</li>
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# proper mixture 1: 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), 0.5 &mu;l Pfu turbo polymerase (KNGiE), 2.5 &mu;l Pfu Turbo Buffer (Fermentas), 2.5 &mu;l MgSO<sub>4</sub> (20 &mu;M; Fermentas), 1 &mu;l DNA matrix, 16.5 &mu;l MQ water
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</ul>
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# proprer mixture 2: 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), 0.5 &mu;l Pfu turbo polymerase (KNGiE), 2.5 &mu;l Pfu Turbo Buffer (Fermentas), 2.5 &mu;l MgSO<sub>4</sub> (20 &mu;M; Fermentas), 2 &mu;l DNA matrix, 15.5 &mu;l MQ water
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<p>1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water</p>
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# Negative control: the same as proper mixture 1, the only distinction is lack of the DNA matrix.
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<ul>
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* Program:
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<li>PCR mixture composition:</li>
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<ol>
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p53 (detailed destription is [https://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009 here])
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<li>proper mixture 1: 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), 0.5 &mu;l Pfu turbo polymerase (KNGiE), 2.5 &mu;l Pfu Turbo Buffer (Fermentas), 2.5 &mu;l MgSO<sub>4</sub> (20 &mu;M; Fermentas), 1 &mu;l DNA template, 16.5 &mu;l MQ water</li>
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<li>proprer mixture 2: 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), 0.5 &mu;l Pfu turbo polymerase (KNGiE), 2.5 &mu;l Pfu Turbo Buffer (Fermentas), 2.5 &mu;l MgSO<sub>4</sub> (20 &mu;M; Fermentas), 2 &mu;l DNA template, 15.5 &mu;l MQ water</li>
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Results:
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<li>Negative control: the same as proper mixture 1, the only distinction is lack of the DNA template.</li>
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</ol></ul>
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*Clean-up the PCR products
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<ul>
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<li>Program:</li>
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Procedure: DNA was purified using the A&A clean-up kit. Detailed procedure is described [http://www.aabiot.com/products/dna_purification/dna_fragments/clean_up/protocol_clean_up.pdf here]
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</ul>
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<br/>
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After clean-up 1 &mu;l of purified PCR product was loaded into gel and photographed. Unfortunatelly reaction 1 (2 &mu;l of DNA matrix) was abortive - there is no product in the gel.
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p53 (detailed destription is <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009">here</a>
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<br/>
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[[image:PCR_reaction-p53_13_07_09.png|thumb|center|300px|verification of PCR reaction and clean-up procedure]]
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<p>Results:</p>
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<ul>
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Task 2:
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<li>Clean-up the PCR products</li>
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</ul>
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*Restriction digest of p53 coding sequence.
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<p>Procedure: 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><p>
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Methods:
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<p>After clean-up 1 &mu;l of purified PCR product was loaded into gel and photographed. Unfortunatelly reaction 1 (2 &mu;l of DNA matrix) was abortive - there is no product in the gel.<p>
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*Control digest using PvuII  
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<br/>
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** Reaction mixture composition: 1 &mu;l purified PCR product, 0.5 &mu;l PvuII (Fermentas), 2 &mu;l Buffer Green (Fermentas), 15.5 &mu;l MQ water
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<center>
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*Digest for subsequent cloning using XbaI
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<img src="https://static.igem.org/mediawiki/2009/2/26/PCR_reaction-p53_13_07_09.png" width="45%" height="45%">
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** Reaction mixture composition: 10 &mu;l purified PCR product, 1 &mu;l XbaI (Fermentas), 5 &mu;l Buffer Tango (Fermentas), 34.5 &mu;l MQ water
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</center>
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* Both reaction were perform in the same condition:
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<p><div style="text-align: center;">verification of PCR reaction and clean-up procedure</div><p>
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Program:
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<br/>
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<p>Task 2:</p>
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digest:
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<ul>
 +
<li>Restriction digest of p53 coding sequence.</li>
 +
</ul>
 +
<p>Methods:</p>
 +
<ul>
 +
<li>Control digest using PvuII</li>
 +
<ul><li>Reaction mixture composition: 1 &mu;l purified PCR product, 0.5 &mu;l PvuII (Fermentas), 2 &mu;l Buffer Green (Fermentas), 15.5 &mu;l MQ water</li></ul>
 +
<li>Digest for subsequent cloning using XbaI</li>
 +
<ul><li>Reaction mixture composition: 10 &mu;l purified PCR product, 1 &mu;l XbaI (Fermentas), 5 &mu;l Buffer Tango (Fermentas), 34.5 &mu;l MQ water</li></ul>
 +
<li>Both reaction were perform in the same condition:</li>
 +
</ul>
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<p>Program:</p>
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<p>digest:</p>
<pre>
<pre>
1. 37&deg;C - 3 hours
1. 37&deg;C - 3 hours
Line 47: Line 59:
3. 4&deg;C - hold
3. 4&deg;C - hold
</pre>
</pre>
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<br/><center>
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[[image:P53_control_digest_13_07_09.png‎|thumb|300px|center|control digest of p53 with PvuII ]]
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<img src="https://static.igem.org/mediawiki/2009/6/63/P53_control_digest_13_07_09.png" heigth="50%" width="50%"><center>
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<div style="text-align: central;">control digest of p53 with PvuII</div>
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Restriction pattern confirm that sequence is correct
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<br/>
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<p><b><Comment</b></p>
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*Purification of digested products via gel-out
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<p>Restriction pattern confirms that sequence is correct</p>
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<ul>
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Procedure:
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<li>Purification of digested products via gel-out</li>
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*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 [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf here]
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</ul>
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<br/>
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*Quantification of amount of p53 DNA after restriction digest:
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<p>Procedure:<p/>
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<ul>
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1 &mu;l of the digest mixture was diluted to 10 &mu;l and loaded into the gel.
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<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>
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<li>Quantification of amount of p53 DNA after restriction digest:</li>
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[[image:P53_PCR_after_digest_13_07_09.png‎ |thumb|center|200px|PCR product after digest loaded into the gel]]
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<ul>
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<p>1 &mu;l of the digest mixture was diluted to 10 &mu;l and loaded into the gel.</p>
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Task 3:
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<br/><center>
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<img src="https://static.igem.org/mediawiki/2009/4/47/P53_PCR_after_digest_13_07_09.png" width="50%" heigth="50%"><center>
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*Cloning p53 coding sequence to pKS plasmid
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<p><div style="text-align: central;">PCR product after digest loaded into the gel</div></p>
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Methods:
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<br/>
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*Ligation mixture composition: 14 &mu;l digested p53, 1.5 &mu;l digested pKS, 5 &mu;l ligation buffer (Invitrogen), 1 &mu;l ligase T4
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<p>Task 3:</p>
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* Duration of ligation was about 12 hours   
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<br/>
 +
<ul>
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<li>Cloning p53 coding sequence to pKS plasmid</li>
 +
</ul>
 +
<p>Methods:</p>
 +
<ul>
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<li>Ligation mixture composition: 14 &mu;l digested p53, 1.5 &mu;l digested pKS, 5 &mu;l ligation buffer (Invitrogen), 1 &mu;l ligase T4</li>
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<li>Duration of ligation was about 12 hours</li>
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<ul>
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</html>  

Revision as of 08:33, 19 July 2009


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Cloning the 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:
    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), 0.5 μl Pfu turbo polymerase (KNGiE), 2.5 μl Pfu Turbo Buffer (Fermentas), 2.5 μl MgSO4 (20 μM; Fermentas), 1 μl DNA template, 16.5 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), 0.5 μl Pfu turbo polymerase (KNGiE), 2.5 μl Pfu Turbo Buffer (Fermentas), 2.5 μl MgSO4 (20 μM; Fermentas), 2 μl DNA template, 15.5 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:

  • Clean-up the PCR products

Procedure: DNA was purified using the A&A clean-up kit. Detailed procedure is described here

After clean-up 1 μl of purified PCR product was loaded into gel and photographed. Unfortunatelly reaction 1 (2 μl of DNA matrix) was abortive - there is no product in the gel.


verification of PCR reaction and clean-up procedure


Task 2:

  • Restriction digest of p53 coding sequence.

Methods:

  • Control digest using PvuII
    • Reaction mixture composition: 1 μl purified PCR product, 0.5 μl PvuII (Fermentas), 2 μl Buffer Green (Fermentas), 15.5 μl MQ water
  • 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
  • Both reaction were perform in the same condition:

Program:

digest:

1. 37°C - 3 hours
2. 80°C - 15 minutes
3. 4°C - hold

control digest of p53 with PvuII

Restriction pattern confirms that sequence is correct

  • Purification of digested products via gel-out

Procedure:

  • 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 here
  • Quantification of amount of p53 DNA after restriction digest:

      • 1 μl of the digest mixture was diluted to 10 μl and loaded into the gel.


      PCR product after digest loaded into the gel


      Task 3:


      • Cloning p53 coding sequence to pKS plasmid

      Methods:

      • Ligation mixture composition: 14 μl digested p53, 1.5 μl digested pKS, 5 μl ligation buffer (Invitrogen), 1 μl ligase T4
      • Duration of ligation was about 12 hours


          • Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])

          Franek


          Tasks:

          • Digestion of [http://partsregistry.org/Part:BBa_R0010 BBa_R0010 - lacI regulated promoter], [http://partsregistry.org/Part:BBa_R0080 BBa_R0080 - AraC regulated promoter] and [http://partsregistry.org/Part:BBa_E0840 E0840 – RBS + GFP + Terminator]
          • Ligation of [http://partsregistry.org/Part:BBa_R0010 lacI regulated promoter] and [http://partsregistry.org/Part:BBa_R0080 AraC regulated promoter] with [http://partsregistry.org/Part:BBa_E0840 E0840]


          Methods:

          • DNA containing [http://partsregistry.org/Part:BBa_R0080 pAraC] and [http://partsregistry.org/Part:BBa_R0010 placI] was digested with SpeI and PstI enzymes. Digestion mix contained 10µl of extracted DNA, 2µl of Fermentas Tango buffer, 0.5µl of each enzyme and water added to obtain 20µl total volume. Both mixes were incubated for 3h at 37°C.
          • DNA containing [http://partsregistry.org/Part:BBa_E0840 E0840] was digested with XbaI and PstI enzymes. Digestion mix contained 26µl of extracted DNA, 3µl of Fermentas Tango buffer, 0.5µl of each enzyme. Two identical mixes created this way were incubated for 3h at 37°C.
          • Enzymes in all 4 mixes were inactivated through incubation at 80°C for 20 min.
          • Two ligation mixes were prepared. Each contained 30µl of [http://partsregistry.org/Part:BBa_R0080 pAraC]/ [http://partsregistry.org/Part:BBa_R0010 placI] digestion mix, 20µl of [http://partsregistry.org/Part:BBa_E0840 E0840] mix, 5µl of dNTPs and 2µl of ligase. Both were left at 16°C for over night incubation.


          Results:

          • Positive selection will be made according to fluorescence under UV light

          Cloning of the mgtc promoter into the pKSII+ plasmid

          Kamil


          Tasks:

          • Plasmid assembly

          Methods (the bulk technique):

          • Plasmid digest mix was prepared as follows: 2μl Tango buffer (Fermentas), 3μl pKSII+ plasmid, 2μl XbaI enzyme, 2μl SmaI enzyme, the solution was topped up with H2O to the final volume of 20 ul.
          • mgtc promoter digest mix was prepared as follows: 2μl Tango buffer (Fermentas), 10μl purified gene, 2μl XbaI enzyme, the solution was topped up with H2O to the final volume of 20 ul.
          • The digest was kept for 3h at 37°C, and then the enzyme was inactivated for 15min. at 80°C.

          • The ligation mix was prepared as follows: both inactivated digests were mixed together and topped with 5μl of 30% PEG, 5μl 10mM ATP, 1,2μl Tango buffer (Fermentas) and 2μl of T4 ligase (Fermentas). The ligation was carried out in 18°C overnight (~18h) and then inactivated for 10min. at 65°C.





            • April
            MTWTFSS
                [http://2009.igem.org/Team:Warsaw/Calendar-Main/1_April_2009 1] [http://2009.igem.org/Team:Warsaw/Calendar-Main/2_April_2009 2] [http://2009.igem.org/Team:Warsaw/Calendar-Main/3_April_2009 3] [http://2009.igem.org/Team:Warsaw/Calendar-Main/4_April_2009 4] [http://2009.igem.org/Team:Warsaw/Calendar-Main/5_April_2009 5]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/6_April_2009 6] [http://2009.igem.org/Team:Warsaw/Calendar-Main/7_April_2009 7] [http://2009.igem.org/Team:Warsaw/Calendar-Main/8_April_2009 8] [http://2009.igem.org/Team:Warsaw/Calendar-Main/9_April_2009 9] [http://2009.igem.org/Team:Warsaw/Calendar-Main/10_April_2009 10] [http://2009.igem.org/Team:Warsaw/Calendar-Main/11_April_2009 11] [http://2009.igem.org/Team:Warsaw/Calendar-Main/12_April_2009 12]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/13_April_2009 13] [http://2009.igem.org/Team:Warsaw/Calendar-Main/14_April_2009 14] [http://2009.igem.org/Team:Warsaw/Calendar-Main/15_April_2009 15] [http://2009.igem.org/Team:Warsaw/Calendar-Main/16_April_2009 16] [http://2009.igem.org/Team:Warsaw/Calendar-Main/17_April_2009 17] [http://2009.igem.org/Team:Warsaw/Calendar-Main/18_April_2009 18] [http://2009.igem.org/Team:Warsaw/Calendar-Main/19_April_2009 19]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/20_April_2009 20] [http://2009.igem.org/Team:Warsaw/Calendar-Main/21_April_2009 21] [http://2009.igem.org/Team:Warsaw/Calendar-Main/22_April_2009 22] [http://2009.igem.org/Team:Warsaw/Calendar-Main/23_April_2009 23] [http://2009.igem.org/Team:Warsaw/Calendar-Main/24_April_2009 24] [http://2009.igem.org/Team:Warsaw/Calendar-Main/25_April_2009 25] [http://2009.igem.org/Team:Warsaw/Calendar-Main/26_April_2009 26]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/27_April_2009 27] [http://2009.igem.org/Team:Warsaw/Calendar-Main/28_April_2009 28] [http://2009.igem.org/Team:Warsaw/Calendar-Main/29_April_2009 29] [http://2009.igem.org/Team:Warsaw/Calendar-Main/30_April_2009 30]
            May
            MTWTFSS
                    [http://2009.igem.org/Team:Warsaw/Calendar-Main/1_May_2009 1] [http://2009.igem.org/Team:Warsaw/Calendar-Main/2_May_2009 2] [http://2009.igem.org/Team:Warsaw/Calendar-Main/3_May_2009 3]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/4_May_2009 4] [http://2009.igem.org/Team:Warsaw/Calendar-Main/5_May_2009 5] [http://2009.igem.org/Team:Warsaw/Calendar-Main/6_May_2009 6] [http://2009.igem.org/Team:Warsaw/Calendar-Main/7_May_2009 7] [http://2009.igem.org/Team:Warsaw/Calendar-Main/8_May_2009 8] [http://2009.igem.org/Team:Warsaw/Calendar-Main/9_May_2009 9] [http://2009.igem.org/Team:Warsaw/Calendar-Main/10_May_2009 10]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/11_May_2009 11] [http://2009.igem.org/Team:Warsaw/Calendar-Main/12_May_2009 12] [http://2009.igem.org/Team:Warsaw/Calendar-Main/13_May_2009 13] [http://2009.igem.org/Team:Warsaw/Calendar-Main/14_May_2009 14] [http://2009.igem.org/Team:Warsaw/Calendar-Main/15_May_2009 15] [http://2009.igem.org/Team:Warsaw/Calendar-Main/16_May_2009 16] [http://2009.igem.org/Team:Warsaw/Calendar-Main/17_May_2009 17]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/18_May_2009 18] [http://2009.igem.org/Team:Warsaw/Calendar-Main/19_May_2009 19] [http://2009.igem.org/Team:Warsaw/Calendar-Main/20_May_2009 20] [http://2009.igem.org/Team:Warsaw/Calendar-Main/21_May_2009 21] [http://2009.igem.org/Team:Warsaw/Calendar-Main/22_May_2009 22] [http://2009.igem.org/Team:Warsaw/Calendar-Main/23_May_2009 23] [http://2009.igem.org/Team:Warsaw/Calendar-Main/24_May_2009 24]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/25_May_2009 25] [http://2009.igem.org/Team:Warsaw/Calendar-Main/26_May_2009 26] [http://2009.igem.org/Team:Warsaw/Calendar-Main/27_May_2009 27] [http://2009.igem.org/Team:Warsaw/Calendar-Main/28_May_2009 28] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/29_May_2009&preload=Team:Warsaw/NotebookPreload&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/30_May_2009&preload=Team:Warsaw/NotebookPreload&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/31_May_2009&preload=Team:Warsaw/NotebookPreload&action=edit 31]
            June
            MTWTFSS
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/1_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/2_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/3_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/4_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/5_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/6_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/7_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 7]
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/8_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/9_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/10_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/11_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/12_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/13_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/14_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 14]
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/15_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 15] [http://2009.igem.org/Team:Warsaw/Calendar-Main/16_June_2009 16] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/17_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 17] [http://2009.igem.org/Team:Warsaw/Calendar-Main/18_June_2009 18] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/19_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/20_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/21_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 21]
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/22_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 22] [http://2009.igem.org/Team:Warsaw/Calendar-Main/23_June_2009 23] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/24_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/25_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 25] [http://2009.igem.org/Team:Warsaw/Calendar-Main/26_June_2009 26] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/27_June_2009&preload=Team:Warsaw/NotebookPreload&action=edit 27] [http://2009.igem.org/Team:Warsaw/Calendar-Main/28_June_2009 28]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/29_June_2009 29] [http://2009.igem.org/Team:Warsaw/Calendar-Main/30_June_2009 30]
            July
            MTWTFSS
                [http://2009.igem.org/Team:Warsaw/Calendar-Main/1_July_2009 1] [http://2009.igem.org/Team:Warsaw/Calendar-Main/2_July_2009 2] [http://2009.igem.org/Team:Warsaw/Calendar-Main/3_July_2009 3] [http://2009.igem.org/Team:Warsaw/Calendar-Main/4_July_2009 4] [http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009 5]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/6_July_2009 6] [http://2009.igem.org/Team:Warsaw/Calendar-Main/7_July_2009 7] [http://2009.igem.org/Team:Warsaw/Calendar-Main/8_July_2009 8] [http://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009 9] [http://2009.igem.org/Team:Warsaw/Calendar-Main/10_July_2009 10] [http://2009.igem.org/Team:Warsaw/Calendar-Main/11_July_2009 11] [http://2009.igem.org/Team:Warsaw/Calendar-Main/12_July_2009 12]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/13_July_2009 13] [http://2009.igem.org/Team:Warsaw/Calendar-Main/14_July_2009 14] [http://2009.igem.org/Team:Warsaw/Calendar-Main/15_July_2009 15] [http://2009.igem.org/Team:Warsaw/Calendar-Main/16_July_2009 16] [http://2009.igem.org/Team:Warsaw/Calendar-Main/17_July_2009 17] [http://2009.igem.org/Team:Warsaw/Calendar-Main/18_July_2009 18] [http://2009.igem.org/Team:Warsaw/Calendar-Main/19_July_2009 19]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/20_July_2009 20] [http://2009.igem.org/Team:Warsaw/Calendar-Main/21_July_2009 21] [http://2009.igem.org/Team:Warsaw/Calendar-Main/22_July_2009 22] [http://2009.igem.org/Team:Warsaw/Calendar-Main/23_July_2009 23] [http://2009.igem.org/Team:Warsaw/Calendar-Main/24_July_2009 24] [http://2009.igem.org/Team:Warsaw/Calendar-Main/25_July_2009 25] [http://2009.igem.org/Team:Warsaw/Calendar-Main/26_July_2009 26]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/27_July_2009 27] [http://2009.igem.org/Team:Warsaw/Calendar-Main/28_July_2009 28] [http://2009.igem.org/Team:Warsaw/Calendar-Main/29_July_2009 29] [http://2009.igem.org/Team:Warsaw/Calendar-Main/30_July_2009 30] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/31_July_2009&preload=Team:Warsaw/NotebookPreload&action=edit 31]
            August
            MTWTFSS
                      [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/1_August_2009&preload=Team:Warsaw/NotebookPreload&action=edit 1] [http://2009.igem.org/Team:Warsaw/Calendar-Main/2_August_2009 2]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/3_August_2009 3] [http://2009.igem.org/Team:Warsaw/Calendar-Main/4_August_2009 4] [http://2009.igem.org/Team:Warsaw/Calendar-Main/5_August_2009 5] [http://2009.igem.org/Team:Warsaw/Calendar-Main/6_August_2009 6] [http://2009.igem.org/Team:Warsaw/Calendar-Main/7_August_2009 7] [http://2009.igem.org/Team:Warsaw/Calendar-Main/8_August_2009 8] [http://2009.igem.org/Team:Warsaw/Calendar-Main/9_August_2009 9]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/10_August_2009 10] [http://2009.igem.org/Team:Warsaw/Calendar-Main/11_August_2009 11] [http://2009.igem.org/Team:Warsaw/Calendar-Main/12_August_2009 12] [http://2009.igem.org/Team:Warsaw/Calendar-Main/13_August_2009 13] [http://2009.igem.org/Team:Warsaw/Calendar-Main/14_August_2009 14] [http://2009.igem.org/Team:Warsaw/Calendar-Main/15_August_2009 15] [http://2009.igem.org/Team:Warsaw/Calendar-Main/16_August_2009 16]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/17_August_2009 17] [http://2009.igem.org/Team:Warsaw/Calendar-Main/18_August_2009 18] [http://2009.igem.org/Team:Warsaw/Calendar-Main/19_August_2009 19] [http://2009.igem.org/Team:Warsaw/Calendar-Main/20_August_2009 20] [http://2009.igem.org/Team:Warsaw/Calendar-Main/21_August_2009 21] [http://2009.igem.org/Team:Warsaw/Calendar-Main/22_August_2009 22] [http://2009.igem.org/Team:Warsaw/Calendar-Main/23_August_2009 23]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/24_August_2009 24] [http://2009.igem.org/Team:Warsaw/Calendar-Main/25_August_2009 25] [http://2009.igem.org/Team:Warsaw/Calendar-Main/26_August_2009 26] [http://2009.igem.org/Team:Warsaw/Calendar-Main/27_August_2009 27] [http://2009.igem.org/Team:Warsaw/Calendar-Main/28_August_2009 28] [http://2009.igem.org/Team:Warsaw/Calendar-Main/29_August_2009 29] [http://2009.igem.org/Team:Warsaw/Calendar-Main/30_August_2009 30]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/31_August_2009 31]
            September
            MTWTFSS
              [http://2009.igem.org/Team:Warsaw/Calendar-Main/1_September_2009 1] [http://2009.igem.org/Team:Warsaw/Calendar-Main/2_September_2009 2] [http://2009.igem.org/Team:Warsaw/Calendar-Main/3_September_2009 3] [http://2009.igem.org/Team:Warsaw/Calendar-Main/4_September_2009 4] [http://2009.igem.org/Team:Warsaw/Calendar-Main/5_September_2009 5] [http://2009.igem.org/Team:Warsaw/Calendar-Main/6_September_2009 6]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/7_September_2009 7] [http://2009.igem.org/Team:Warsaw/Calendar-Main/8_September_2009 8] [http://2009.igem.org/Team:Warsaw/Calendar-Main/9_September_2009 9] [http://2009.igem.org/Team:Warsaw/Calendar-Main/10_September_2009 10] [http://2009.igem.org/Team:Warsaw/Calendar-Main/11_September_2009 11] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/12_September_2009&preload=Team:Warsaw/NotebookPreload&action=edit 12] [http://2009.igem.org/Team:Warsaw/Calendar-Main/13_September_2009 13]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/14_September_2009 14] [http://2009.igem.org/Team:Warsaw/Calendar-Main/15_September_2009 15] [http://2009.igem.org/Team:Warsaw/Calendar-Main/16_September_2009 16] [http://2009.igem.org/Team:Warsaw/Calendar-Main/17_September_2009 17] [http://2009.igem.org/Team:Warsaw/Calendar-Main/18_September_2009 18] [http://2009.igem.org/Team:Warsaw/Calendar-Main/19_September_2009 19] [http://2009.igem.org/Team:Warsaw/Calendar-Main/20_September_2009 20]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/21_September_2009 21] [http://2009.igem.org/Team:Warsaw/Calendar-Main/22_September_2009 22] [http://2009.igem.org/Team:Warsaw/Calendar-Main/23_September_2009 23] [http://2009.igem.org/Team:Warsaw/Calendar-Main/24_September_2009 24] [http://2009.igem.org/Team:Warsaw/Calendar-Main/25_September_2009 25] [http://2009.igem.org/Team:Warsaw/Calendar-Main/26_September_2009 26] [http://2009.igem.org/Team:Warsaw/Calendar-Main/27_September_2009 27]
            [http://2009.igem.org/Team:Warsaw/Calendar-Main/28_September_2009 28] [http://2009.igem.org/Team:Warsaw/Calendar-Main/29_September_2009 29] [http://2009.igem.org/Team:Warsaw/Calendar-Main/30_September_2009 30]
            October
            MTWTFSS
                  [http://2009.igem.org/Team:Warsaw/Calendar-Main/1_October_2009 1] [http://2009.igem.org/Team:Warsaw/Calendar-Main/2_October_2009 2] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/3_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/4_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 4]
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/5_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/6_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/7_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/8_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/9_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/10_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/11_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 11]
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/12_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 12] [http://2009.igem.org/Team:Warsaw/Calendar-Main/13_October_2009 13] [http://2009.igem.org/Team:Warsaw/Calendar-Main/14_October_2009 14] [http://2009.igem.org/Team:Warsaw/Calendar-Main/15_October_2009 15] [http://2009.igem.org/Team:Warsaw/Calendar-Main/16_October_2009 16] [http://2009.igem.org/Team:Warsaw/Calendar-Main/17_October_2009 17] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/18_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 18]
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/19_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/20_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/21_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/22_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/23_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/24_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/25_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 25]
            [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/26_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/27_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/28_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/29_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/30_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/31_October_2009&preload=Team:Warsaw/NotebookPreload&action=edit 31]
Retrieved from "http://2009.igem.org/Team:Warsaw/Calendar-Main/13_July_2009"