Team:Warsaw/Calendar-Main/10 October 2008

Visit in US Embassy Visas have been accorded to the whole team.

Preparation of alpha_linker under PT7 (BBa_K103019) Michał K., Piotr  Clean-up of overnight digest reaction.  Digest of pSB1A3 carrying &Delta;A (BBa_K103003) with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP </li> Gel electrophoresis and gel-out</a> of proper band - 2200 bp. Fig. 1</a>. </li> Ligation</a> of digested <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> with alpha_linker under PT7 (BBa_K103019)</a> fragment (1 hr).</li>  Transformation</a> of TOP10</a> with above ligation.</li>  Tranformants plating on LB with ampicillin. </li> </ol>

<img src="http://2008.igem.org/wiki/images/f/fd/Go_10_10_2008.jpg"> Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha 1. Marker 2. PCR to obtain alpha_linker under PT7 (BBa_K103019) 3. PCR to obtain AID under pBAD/araC (BBa_K103002) 4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of omega_linker under PT7 (BBa_K103020)</a> Michał K., Piotr <ol> Ligation</a> of digested pSB2K3</a> vector from (<A href=http://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008>30 September</a>) with omega_linker under PT7 (BBa_K103020)</a> fragment (1 hr).</li>  <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with above ligation.</li> <li> Tranformants plating on LB with kanamycin. </li></ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a> Michał K., 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://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site).</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 BamHI (BamHI buffer) - proper clones found.</li> <li>Temperature gradient <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> (with removed EcoRI site) plasmid using

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AraCl">AraCl</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpHSNP">AIDP_HindSpeNotPst</a>

primers (annealing temperature 40 - 60 &deg;C; elongation length 2.30 min) to optimize conditions for obtaining <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a>. </li> <li>Gel electrophoresis. Best annealing temperature (45 &deg;C) chosen. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig2">Fig. 2</a>.</li>

<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> (with removed EcoRI site) plasmid using

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AraCl">AraCl</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpHSNP">AIDP_HindSpeNotPst</a>

primers (annealing temperature 45 &deg;C; elongation length 2.30 min) to obtain <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a>fragment. </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 1800 bp. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig1">Fig. 1</a>. </li> <li><a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of isolated 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 digested PCR product. </li></ol>

<img src="http://2008.igem.org/wiki/images/f/fe/Grad_arac_09_10_2008.jpg"></a> Fig. 2.Gradient PCR products for AID under pBAD/araC 1 -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 40&deg;C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 60&deg;C (the highest temperature of gradient).

<img src="http://2008.igem.org/wiki/images/f/fd/Go_10_10_2008.jpg"> Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha 1. Marker 2. PCR to obtain alpha_linker under PT7 (BBa_K103019) 3. PCR to obtain AID under pBAD/araC (BBa_K103002) 4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha

Preparation of <a href=http://partsregistry.org/Part:BBa_K103017>OmpA_linker_alpha_linker under Plac (BBa_K103017)</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/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP+link10+homo2">AlphaP+link10+homo2</a> primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha fragment. </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 600 bp. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/10_October_2008#fig1">Fig. 1</a>. </li>

</ol>

<img src="http://2008.igem.org/wiki/images/f/fd/Go_10_10_2008.jpg"></a> Fig. 1.PCR to obtain alpha, PCR to obtain ara_pbad_aid, digest of pACYC177+OmpA_omega_DeltaA_alpha 1. Marker 2. PCR to obtain alpha_linker under PT7 (BBa_K103019) 3. PCR to obtain AID under pBAD/araC (BBa_K103002) 4. EcoRI/BcuI digest of pACYC177+OmpA_omega_DeltaA_alpha