Team:Warsaw/Calendar-Main/29 July 2008

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<p>1. Separete transformant colonies (tranformation from previous day) inoculated to liquid LB with kanamycin. <br>
 
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2. Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.</p>
 
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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr</h4>
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<p><A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BZ-alpha>pET15b+Z_alpha</A> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2Bhis%2BZ%2Bomega>pET15b+Z_omega</a> in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain inoculated in liquid LB with chloramphenicol and ampicillin.
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<h3>Cloning of omega_&Delta;A DNA fragment to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a></h3><h4>Michał K.</h4>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a> from culture inoculated on previous day.</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a>  with SacI and NotI (BamHI buffer).</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> (SacI and NotI) and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP) of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>. </li>
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<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 4300 bp (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>) and 550 bp (omega_&Delta;A) bands (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_July_2008#fig1">Fig. 1.</a>).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of DNA fragments from 2. and 3. (1 hr).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation.</li>
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<li>Transformants plating on LB + kanamycin.</li></ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/6/6a/Po_go.jpg" width=200/></a> <var><b>Fig. 1. </b> SacI/NotI digests of isolated plasmids<br>
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1. Marker<br>
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2. digested pKS_omega_&Delta;A<br>
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3. digested pACYC_Omp_alpha<br></var>
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Sprawdzanie czy omp_omega_A_alfa daje oporność na AP Piotr
 
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Zaszczepienie omp_omega_A_alfa z rużnych stężeń IPTG: 0; 0,1 ; 0,25 ; 0,5; 0,75; 1 do takich samych stężeń IPTG, ale z rużnymi stężeniami AP (25;50;75;100) w stosunku 1:50
 
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wieczorem mieżenie OD : tabelka
 
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Zaszczepienie omp_omega_A_alfa na rużnych stężeń IPTG: 0; 0,1 ; 0,25 ; 0,5; 0,75 (do powtużenia )
 
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Sprawdzenie ekspresji omp_omega_A_alfa i omp_A_alfa Piotr
 
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Zwirowanie, zawieszenie, dodanie buforu lizującego (skład), zagotowanie, nałożenie na żel poliakryl, transfer na nitrocelulozę, blokowanie, przeciwciała anty-A, płukanie, przeciwciała anty-królik, wywołanie BCIP i NBT (omp_omega_A_alfa, z i bez indukcji, omp_A_alfa)
 
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(Zdjęcie żelu)
 
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Latest revision as of 16:55, 28 October 2008

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Purification of proteins: Z-alpha and Z-omega

Piotr

pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain inoculated in liquid LB with chloramphenicol and ampicillin.

Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

  1. Isolation of pKS+omega_ΔA from culture inoculated on previous day.
  2. Digest of pKS+omega_ΔA with SacI and NotI (BamHI buffer).
  3. Digest (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
  4. Gel electrophoresis and gel-out of 4300 bp (pACYC177+OmpA_alpha) and 550 bp (omega_ΔA) bands (Fig. 1.).
  5. Ligation of DNA fragments from 2. and 3. (1 hr).
  6. Transformation of E. coli TOP10 strain with ligation.
  7. Transformants plating on LB + kanamycin.

Fig. 1. SacI/NotI digests of isolated plasmids
1. Marker
2. digested pKS_omega_ΔA
3. digested pACYC_Omp_alpha

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