Team:Warsaw/Calendar-Main/6 October 2008

Preparation of linker_alpha (BBa_K103009) Michał K.  Colony PCR with LinLSXNE and AlphaPSpe

primers on colonies from plates with transformations pSB1A3+ linker_alpha (BBa_K103009) (annealing temperature - 55&deg;C,60 s of elongation step). No visible bands for proper product (Fig. 1).</li>

<li>Inoculation of some colonies which grown on plate - pSB2K3</a> + linker_alpha (BBa_K103009)</a> to liquid LB + kanamycin. </li></ol>

<img src="http://2008.igem.org/wiki/images/6/66/Kolonijny_alfa.jpg"></a> Fig. 1.Colony PCR with LinLSXNE and AlphaPSpe. Lack of expected 400 bp product.

Preparation of linker_omega (BBa_K103013)</a> Michał K. Inoculation of some colonies which grown on plate - pSB2K3</a> + linker_omega (BBa_K103013)</a> to liquid LB + kanamycin.

Preparation of OmpA-linker-omega-linker (BBa_K103016)</a> Michał K. <ol> <li> Colony PCR</a> with OmpaL_N</a> and OmpaP_link</a>

primers on colonies from plates with transformations pACYC177</a> + OmpA-linker-omega-linker (BBa_K103016)</a> (annealing temperature - 55&deg;C,90 s of elongation step). No visible bands for proper product (Fig. 2</a>).</li> <li>Inoculation of some colonies which grown on plate transformation: pACYC177</a> + OmpA-linker-omega-linker (BBa_K103016)</a> to liquid LB + kanamycin. </li></ol>

<img src="http://2008.igem.org/wiki/images/c/ce/Kolonijny_ompa_01_10_2008.jpg"></a> Fig. 2.Colony PCR with OmpaL_N and OmpaP_link. Lack of expected 350 bp product.

Preparation of vector for pT7 constructs Michał K. Overnight selfligation</a> of pET15b+OmpA_omega</a> (with removed XbaI).

Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Michał K. Overnight <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> (from <a href=http://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008>1 October</a>) + <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> (without EcoRI site).

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/Team:Warsaw/protocols#digest">Digest</a> of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> plasmid with XbaI and PstI (Tango buffer). <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li> <li> Gel electrophoresis 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 band - 2200 bp. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/6_October_2008#fig3">Fig. 3</a>.</li> </ol>

<img src="http://2008.igem.org/wiki/images/4/49/Go_05_10_2008.jpg"></a> Fig. 3.XbaI/PstI digest of pSB1A3. 1. Marker 2. XbaI/PstI digest pSB1A3