Team:Warsaw/Calendar-Main/1 October 2008

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</a> of proper bands: 4500 bp - pSB2K3</a> and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector)</a>. Fig. 1</a>.</li> </ol>

<img src="http://2008.igem.org/wiki/images/2/23/Go_01_10_2008.jpg" width=150></a> 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)</a> Michał K. Inoculation of some colonies from pSB1A3</a>+Z(BBa_K103004)</a> plate to LB with ampicillin.

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Michał K. <ol> Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of BBa_K103018</a> fragment. Ligation for 1 hr. </li> PCR</a> on ligation of OmpA_linker_omega_linker under Plac (BBa_K103018)</a> fragment using PlacL_XNE</a> and LinP_BS</a> primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain BBa_K103018</a> fragment without internal EcoRI site (Przanowski's method of removing internal restriction site). </li> <li> Gel electrophoresis of PCR product 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 (BBa_K103018 - 1200 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008#fig2">Fig. 2</a>.</li> </ol>

<img src="http://2008.igem.org/wiki/images/9/9d/Go2_01_10_2008.jpg" width=150> 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 <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#pcr">PCR</a> on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> plasmid using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AIDl">AIDl</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpSNP">AIDP_SpeNotPst</a> 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>

<li> Gel electrophoresis of PCR product 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 (AID(BBa_K103001) - 600 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008#fig2">Fig. 2</a>.</li> <li><a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of PCR product with XbaI and PstI (Tango buffer).</li>

<li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of above digest reaction. </li> </ol>

<img src="http://2008.igem.org/wiki/images/9/9d/Go2_01_10_2008.jpg"></a> Fig. 2. PCR to obtain AID and OmpA_linker_omega_linker under Plac 1. Marker 2. AID 3. OmpA_linker_omega_linker under Plac