Team:Warsaw/Calendar-Main/22 May 2008

From 2008.igem.org

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<h4>Piotr:</h4>
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Design of primers for sequencing something we want from pMPM-T5<br>
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<h3>Rifampicin test #2</h3>
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<h4>Piotr</h4>
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<p>Overnight inoculation and induction:<br>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">Top10</a><br>
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and <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">Top10</a> carrying:<br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5-AID</a> + 0.01% L-Ara<br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99a-AID</a> + 0.5 mM IPTG<br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5-AID</a><br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99a-AID</a><br>
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<h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPMT5-AID+T7</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPMT5+AID-T7</a></h3>
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<h4>Piotr, Michał K.</h4>
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<ol><li>
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Design of primers for sequencing something we want from pMPM-T5.<br>
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Primers:<br>
Primers:<br>
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<h4>Michał K.:</h4>
 
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - translation fusion: AID + T7 RNA-polymerase<br>
 
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Primers: <br>
 
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a></li>
 
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a></li>
 
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPM-T5 with transcriptional fusion</a>.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPM-T5 with translational fusion</a..</li>
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Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion <br>
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<li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPM-T5 with transcriptional fusion</a> with EcoRI (EcoRI buffer).</li>
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Elongation temperature : 73&deg;C <br>
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<li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPM-T5 with translational fusion</a> with BamHI (BamHI buffer).</li>
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Elongation time: 4 minutes <br>
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20 cycles <br></li>
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<li> Gel electrophoresis (no proper colonies found). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/22_May_2008#fig1">Fig. 1</a>.</li>
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<li> Gel electrophoresis and isolation of proper band (3300 bp).</li></ol></p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/8/87/Aid_fusion_digests_WAW.jpg"width=300/></a>
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<var><b>Fig. 1.</b> 1-DNA ladder;<br>
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2 and 3-hypothetical pMPM-T5 with transcriptional fusion digested with EcoRI (2 clones); <br>
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4-pMPM-T5-AID  digested with EcoRI; <br>
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5-hypothetical pMPM with translational fusion digested with BamHI; <br>
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6-pMPM-T5-AID digested with BamHI.</var>
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Latest revision as of 14:07, 26 October 2008

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Rifampicin test #2

Piotr

Overnight inoculation and induction:
Top10
and Top10 carrying:
pMPM-T5-AID + 0.01% L-Ara
pTrc99a-AID + 0.5 mM IPTG
pMPM-T5-AID
pTrc99a-AID

Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

Piotr, Michał K.

  1. Design of primers for sequencing something we want from pMPM-T5.
    Primers:

  2. Isolation of hypothetical pMPM-T5 with transcriptional fusion.
  3. Isolation of hypothetical pMPM-T5 with translational fusion
  4. Control digest of pMPM-T5 with transcriptional fusion with EcoRI (EcoRI buffer).
  5. Control digest of pMPM-T5 with translational fusion with BamHI (BamHI buffer).
  6. Gel electrophoresis (no proper colonies found). Fig. 1.
    7. Fig. 1. 1-DNA ladder;
    2 and 3-hypothetical pMPM-T5 with transcriptional fusion digested with EcoRI (2 clones);
    4-pMPM-T5-AID digested with EcoRI;
    5-hypothetical pMPM with translational fusion digested with BamHI;
    6-pMPM-T5-AID digested with BamHI.

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