Team:Warsaw/Calendar-Main/9 May 2008

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<html><h3>Preparation of pMPMT5+AID construct</h3>
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<html><h3>Preparation of pMPMT5+AID construct<br>
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<h4>Michał K.:</h4>
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Michał K.</h3>
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<li> DNA electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation of DNA</a> encoding AID from proper band (600 bp).</li>
<li> DNA electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation of DNA</a> encoding AID from proper band (600 bp).</li>
<img src="https://static.igem.org/mediawiki/2008/8/89/Gel_out_aid_WAW.jpg" width=300/>
<img src="https://static.igem.org/mediawiki/2008/8/89/Gel_out_aid_WAW.jpg" width=300/>
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<h3>1-DNA ladder; 2-pTRC99a-AID digested with NcoI and HindIII</h3>
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<em>1-DNA ladder; 2-pTRC99a-AID digested with NcoI and HindIII</em>
<li> Restriction <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-omega>pMPM-T5</a> with HindIII and NcoI (1x Tango buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP).</li>
<li> Restriction <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-omega>pMPM-T5</a> with HindIII and NcoI (1x Tango buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP).</li>
<li> DNA electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-omega>pMPM-T5</a> vector from proper band (4250 bp).</li>
<li> DNA electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-omega>pMPM-T5</a> vector from proper band (4250 bp).</li>
<img src="https://static.igem.org/mediawiki/2008/0/0f/Gel_out_pmpm_WAW.jpg" width=300/>
<img src="https://static.igem.org/mediawiki/2008/0/0f/Gel_out_pmpm_WAW.jpg" width=300/>
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<h3>1-DNA ladder; 2-pMPM-T5 digested with NcoI and HindIII</h3>
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<em>1-DNA ladder; 2-pMPM-T5 digested with NcoI and HindIII</em>
<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of isolated DNA fragments. </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of isolated DNA fragments. </li>

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Preparation of pMPMT5+AID construct
Michał K.

  1. Restriction digest of pTrc99A-AID with HindIII and NcoI (1x Tango buffer).
  2. DNA electrophoresis and isolation of DNA encoding AID from proper band (600 bp).
    3. 1-DNA ladder; 2-pTRC99a-AID digested with NcoI and HindIII
  3. Restriction digest of pMPM-T5 with HindIII and NcoI (1x Tango buffer), dephosphorylation (CIAP).
  4. DNA electrophoresis and isolation of pMPM-T5 vector from proper band (4250 bp).
    5. 1-DNA ladder; 2-pMPM-T5 digested with NcoI and HindIII
  5. Electrophoresis to estimate the concentration of isolated DNA.
  6. Ligation of isolated DNA fragments.






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