Team:Warsaw/Calendar-Main/1 October 2008

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Preparation of vectors for Biobricks

Michał K., Piotr

Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
Gel elctrophoresis and gel-out of proper band: ??????.

Preparation of BioBricks

Michał K.

Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of pLac_OmpA_omega fragment. Ligation for 1 hr.
PCR on pMPMT5+AID plasmid using AIDl and AIDP_HindSpeNotPst primers (annealing temperature 58 °C; elongation length 60s) to obtain AID fragment.
PCR on ligation of pLac_OmpA_omega fragment using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain pLac_OmpA_omega fragment without EcoRI.
Gel electrophoresis of PCR products and gel-out of proper bands (AID - 600 bp and pLac_Omp_omega_ - 1200 bp).
Inoculation of some colonies from pSB1A3+Z(BBa_K103004) plate to LB with ampicillin.

@@ Line 3: / Line 3: @@
 <html>
+<h3>Preparation of vectors for Biobricks</h3>
+<h4>Michał K., Piotr</h4>
+<ol>
+<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>
+<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>
+<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> </ol>
 <h3>Preparation of BioBricks</h3>
 <h4>Michał K.</h4>
@@ Line 18: / Line 29: @@
 <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>
-<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>
-<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>
-<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>
 <li>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.</li></ol>