Team:Warsaw/Calendar-Main/25 September 2008

MutD5 testing Emilia Isolation of plasmid from culture inoculated on previous day. Preparation of samples to sequencing.

 Mutagenesis of protein A Paweł

Treatment of mutageneses as on 23rd September.

Preparation of alpha_A construct Antoni   PCR on alpha_linker and linker_A with AlphaL+SacI and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> primers and 10% DMSO (30 cycles, elongation 60 s, annealing temperature 72&deg;C). </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 band (1000 bp).</li> </ol>

Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> Piotr, Michał K.  Colony <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL_BNXNE">AL_BNXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>

primers on colonies from plates with transformations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> + <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A</a>. No products visible after gel electrophoresis. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig1">Fig. 1</a>.</li> Inoculation of some colonies from plate to LB with ampicillin.</li></ol>

<img src="http://2008.igem.org/wiki/images/b/bf/Kolonijny_25_09.jpg" width=300/></a> Fig. 1. Colony PCR to obtain pSB1A3 + ΔA 1. Marker 2-13. PCR on various colonies

Preparation of <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a> Michał K.   <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+Nde">AlphaL+Nde</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPlinkSac">AlphaPlinkSac</a> primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha_linker fragment. </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 band (alpha_linker - 600 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2</a>.</li>

<a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with NdeI and SacI (BamHI buffer). </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/8/8b/Go_25_09.jpg" width=300/> Fig. 2. Results of PCR to obtain alpha_linker and omega_linker: 1. Marker 2. alpha_link PCR 3. omega_link PCR

Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> Michał K. 

<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#OmegLNde">OmegLNde</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 45s) to obtain omega_linker fragment. </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 band (omega_linker - 350 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2</a>.</li>

<a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#Digest">Digest</a> of purified PCR product with NdeI and SacI (BamHI buffer). </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/8/8b/Go_25_09.jpg" width=300/></a> Fig. 2. Results of PCR to obtain alpha_linker and omega_linker: 1. Marker 2. alpha_link PCR 3. omega_link PCR