Team:Warsaw/Calendar-Main/1 October 2008

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<h3>Preparation of BioBricks</h3>
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<h3>Preparation of vectors for Biobricks</h3>
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<h4>Michał K., Piotr</h4>
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<ol>
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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 <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a> plasmids.</li>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of isolated plasmids with EcoRI and BcuI (BamHI buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmids.</li>
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<li>Gel elctrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands: 4500 bp - <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and 3000 bp - <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a>. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008#fig1">Fig. 1</a>.</li> </ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/2/23/Go_01_10_2008.jpg" width=150></a>
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<var><b>Fig. 1.</b>Digest of pSB2K3 and BBa_I739204 with EcoRI and BcuI<br>
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1. Marker<br>
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2. Digested pSB2K3<br>
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3. Digested BBa_I739204<br></var>
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<h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a></h3>
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<h4>Michał K.</h4>
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<p>Inoculation of some colonies from <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a> plate to LB with ampicillin.</p>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a></h3>
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<h4>Michał K.</h4>
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<ol><li> Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> fragment. Ligation for 1 hr. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on ligation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> fragment using
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PlacL_XNE">PlacL_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
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primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> fragment without internal EcoRI site (Przanowski's method of removing internal restriction site). </li>
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<li> Gel electrophoresis of PCR product and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (BBa_K103018 - 1200 bp).
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<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008#fig2">Fig. 2</a>.</li>
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</ol>
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<p class="hide"><br>
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<img src="https://static.igem.org/mediawiki/2008/9/9d/Go2_01_10_2008.jpg" width=150>
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<var><b>Fig. 2.</b> PCR to obtain AID and OmpA_linker_omega_linker under Plac<br>
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1. Marker<br>
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2. AID<br>
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3. OmpA_linker_omega_linker under Plac<br> </var>
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</p>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol>
<ol>
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<li> Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of pLac_OmpA_omega fragment. Ligation for 1 hr. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> plasmid using
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> plasmid using
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDl">AIDl</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDP_HindSpeNotPst">AIDP_HindSpeNotPst</a>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDl">AIDl</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpSNP">AIDP_SpeNotPst</a>
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primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain AID fragment. </li>
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primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a> fragment. </li>
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 +
 
 +
 
 +
 
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<li> Gel electrophoresis of PCR product and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (AID(BBa_K103001) - 600 bp).
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<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008#fig2">Fig. 2</a>.</li>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of PCR product with XbaI and PstI (Tango buffer).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on ligation of pLac_OmpA_omega fragment using
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PlacL_XNE">PlacL_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
 
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primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain pLac_OmpA_omega fragment without EcoRI. </li>
 
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<li> Gel electrophoresis of PCR products and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (AID - 600 bp and pLac_Omp_omega_ - 1200 bp).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of above digest reaction. </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 HQs(??????) plasmids.</li>
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</ol>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of isolated plasmids with EcoRI and BcuI (BamHI buffer). </li>
 
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<li>Gel elctrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band: ??????. </li>  
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/9/9d/Go2_01_10_2008.jpg"></a>
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<var><b>Fig. 2.</b> PCR to obtain AID and OmpA_linker_omega_linker under Plac<br>
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1. Marker<br>
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2. AID<br>
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3. OmpA_linker_omega_linker under Plac<br></var>
</html>
</html>

Latest revision as of 20:39, 28 October 2008

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Preparation of vectors for Biobricks

Michał K., Piotr

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
  3. Gel elctrophoresis and gel-out of proper bands: 4500 bp - pSB2K3 and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector). Fig. 1.
Fig. 1.Digest of pSB2K3 and BBa_I739204 with EcoRI and BcuI
1. Marker
2. Digested pSB2K3
3. Digested BBa_I739204

Preparation of Z(BBa_K103004)

Michał K.

Inoculation of some colonies from pSB1A3+Z(BBa_K103004) plate to LB with ampicillin.

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

  1. Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of BBa_K103018 fragment. Ligation for 1 hr.
  2. PCR on ligation of OmpA_linker_omega_linker under Plac (BBa_K103018) fragment using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain BBa_K103018 fragment without internal EcoRI site (Przanowski's method of removing internal restriction site).
  3. Gel electrophoresis of PCR product and gel-out of proper bands (BBa_K103018 - 1200 bp). Fig. 2.


Fig. 2. PCR to obtain AID and OmpA_linker_omega_linker under Plac
1. Marker
2. AID
3. OmpA_linker_omega_linker under Plac

Preparation of AID(BBa_K103001)

Michał K.

  1. PCR on pMPMT5+AID plasmid using AIDl and AIDP_SpeNotPst primers (annealing temperature 58 °C; elongation length 60s) to obtain AID(BBa_K103001) fragment.
  2. Gel electrophoresis of PCR product and gel-out of proper bands (AID(BBa_K103001) - 600 bp). Fig. 2.
  3. Digest of PCR product with XbaI and PstI (Tango buffer).
  4. Clean-up of above digest reaction.
Fig. 2. PCR to obtain AID and OmpA_linker_omega_linker under Plac
1. Marker
2. AID
3. OmpA_linker_omega_linker under Plac