Team:Warsaw/Calendar-Main/9 October 2008

Preparation of vector for pT7 constructs Michał K.  Isolation of plasmids from cultures inoculated on previous day - pET15b+OmpA_omega (with removed XbaI site). Control digest of isolated pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found. Fig. 1. Digest</a> of pET15b+OmpA_omega</a> without XbaI plasmid with NdeI and SacI (BamHI buffer), dephosphorylation</a> with CIAP </li> Gel electrophoresis and gel-out</a> of proper band - 6000 bp. Fig. 2</a>. </li>

<img src="http://2008.igem.org/wiki/images/7/77/Traw_petxba_omp_07_10_2008_na_9_10.jpg"></a> Fig. 1.XbaI/BamHI digests of pET15b+OmpA_omega 1. Marker 2-9. XbaI/BamHI digests of pET15b+OmpA_omega

<img src="http://2008.igem.org/wiki/images/d/d3/Gelout_pET_ompa_omega.jpg"/></a> Fig. 2. Digests of pET15b_OmpA_omega without XbaI 1. DNA ladder 2. pET15b_OmpA_omega without XbaI digested with NdeI and SacI

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Preparation of alpha_linker under PT7 (BBa_K103019)</a> Michał K. <ol> Ligation</a> of digested pET15b vector (from <A href=http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=5&arg0=3_October_2008&arg1=6_October_2008&arg2=7_October_2008&arg3=8_October_2008&arg4=9_October_2008&name=Preparation%20of%20vector%20for%20pT7%20constructs>Preparation of vector for pT7 constructs</a>) with alpha_linker fragment(from <A href=http://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008>25 September</a>) (1 hr).</li>

PCR</a> on above ligation using pETt7L_XNE</a> and AlphaPlinkSac</a> primers (annealing temperature 58 &deg;C; elongation length 120s) to obtain alpha_linker under PT7 (BBa_K103019)</a>fragment. </li>  Gel electrophoresis of PCR products and gel-out</a> of proper bands (<a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a> - 800 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/9_October_2008#fig3">Fig. 3</a>.</li>

<li>Overnight <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified PCR product EcoRI and SacI (BamHI buffer). </li> </ol>

<img src="http://2008.igem.org/wiki/images/2/2a/Go2_08_10_2008.jpg" width=200></a> Fig. 3. PCR to obtain pT7_alpha_link and pT7_omega_link 1. Marker 2. PCR to obtain alpha_linker under pT7 3. PCR to obtain omega_linker under pT7

Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> Michał K. <ol> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_linker fragment(from <A href=http://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008>30 September</a>) (1 hr).</li> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on above ligations using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#PETt7L_XNE">pETt7L_XNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 58 &deg;C; elongation length 120s) to obtain <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> fragments </li> <li> Gel electrophoresis of PCR products and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (pT7_omega_ - 600 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/9_October_2008#fig3">Fig. 3</a>.</li>

<li>Overnight <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified PCR product with EcoRI and BcuI (BamHI buffer). </li> </ol>

<img src="http://2008.igem.org/wiki/images/2/2a/Go2_08_10_2008.jpg" width=200></a> Fig. 3. PCR to obtain pT7_alpha_link and pT7_omega_link 1. Marker 2. PCR to obtain alpha_linker under pT7 3. PCR to obtain omega_linker under pT7

Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Piotr <ol><li><a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> +<a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> (without internal EcoRI site).</li>

<li>Control <a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> with EcoRI and PstI (Orange buffer) proper clones found.</li></ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a> Michał K.

<ol> <li><a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmids from cultures inoculated on previous day <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a>.</li> <li>Control <a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/9_October_2008#fig4">Fig. 4</a>.</li>

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<img src="http://2008.igem.org/wiki/images/6/66/Traw_aid_08_10_2008.jpg" width=180></a> Fig.4.Control EcoRI/PstI digests of pSB1A3+AID 1. Marker 2-5. Control EcoRI/PstI digests of pSB1A3+AID

Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a> Piotr

Inoculation of colonies from plate with ligation of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site) to liquid LB + tetracycline.