Team:Warsaw/Calendar-Main/2 October 2008

Preparation of linker_alpha (BBa_K103009) Michał K. Overnight ligation of isolated DNA fragments: pSB2K3 + linker_alpha (BBa_K103009).

Preparation of linker_omega (BBa_K103013) Michał K. Overnight ligation of isolated DNA fragments: pSB2K3 + linker_omega (BBa_K103013).

Preparation of OmpA-linker-omega-linker (BBa_K103016)</a> Michał K. Overnight ligation</a> of DNA fragments isolated  on 30 September</a> : pACYC177</a> + OmpA-linker-omega-linker (BBa_K103016)</a>.

Preparation of OmpA-linker (BBa_K103006)</a> Michał K. <ol><li> Colony PCR</a> with OmpaL_N</a> and OmpaP_link</a>

primers on colonies from plates with transformations pSB1A3</a>+OmpA-linker (BBa_K103006)</a> (annealing temperature - 55&deg;C,45 s of elongation step). </li>

<li> Gel electrophoresis (Fig. 1</a>).</li> <li> Confirmed transformant colonies inoculated to liquid LB with ampicillin.</li> </ol>

<img src="http://2008.igem.org/wiki/images/9/92/Kolonijny_rfp_omp_03_10_2008.jpg"></a> Fig. 1.Colony PCR with OmpaL_N and OmpaP_link. 1. Marker 4, 14, 19, 20, 24. Proper bands visible.

Preparation of Z(BBa_K103004)</a> Michał K. <ol><li>Isolation</a> of plasmids from culture inoculated on previous day (pSB1A3</a>+Z(BBa_K103004)</a>).</li> <li>Control <a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found (<li> Gel electrophoresis (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/2_October_2008#fig2">Fig. 2</a>).</li></ol>

<img src="http://2008.igem.org/wiki/images/6/6f/Traw_02_10_2008.jpg"></a> Fig. 2.Control digest of pSB1A3+Z(BBa_K103004) 1. Marker 3-5. Clones carrying BBa_K103004

Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Michał K. <ol> <li> <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> fragment with EcoRI and BcuI (BamHI buffer). </li> <li> Gel electrophoresis and <a href="http://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 1200 bp. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/2_October_2008#fig3">Fig. 3</a>.</li>

</ol> <img src="http://2008.igem.org/wiki/images/5/54/Go_02_10_2008_na_2_10.jpg"></a> Fig. 3.Digest of OmpA_linker_omega_linker under Plac (BBa_K103018). 1. Marker 2. OmpA_linker_omega_linker under Plac (BBa_K103018)