Team:Warsaw/Calendar-Main/31 July 2008
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+ | <h3> Cloning of truncated fragment of protein A (ΔA)</h3> | ||
+ | <h4> Michał K.</h4> | ||
+ | <p><ol> | ||
+ | <li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> strain with ligation products (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYC177+OmpA_alpha + ΔA</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + ΔA</a>). </li> | ||
+ | <li> Transformants plating on LB + kanamycin. </li></ol></p> | ||
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+ | <h3>Cloning of omega_ΔA 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> | ||
+ | <p> | ||
+ | <ol><li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008">previous day</a>. </li> | ||
+ | <li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with BamHI and SacI (BamHI buffer). </li><li> Gel electrophoresis - proper clones founded (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig1">Fig. 1.</a>). </li></ol> | ||
+ | </p> | ||
+ | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/2/2e/31_july.jpg" width=250/></a> <var><b>Fig. 1. </b>Control SacI/BamHI digests of isolated plasmids<br> | ||
+ | 1-4. digested plasmids pACYC177 + OmpA-omega-ΔA-alpha<br> | ||
+ | 5. Marker<br></var> | ||
+ | <h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr, Emilia</h4> | ||
+ | <p><ol><li>Samples were resuspended in PBS, sonicated and centrifuged.</li> | ||
+ | <li>Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min. </li> | ||
+ | <li>Lysates loaded on 12% poliacrylamide gel (amount relating to 100 μl of OD=1.0 culture).</li> | ||
+ | <li>Gel stained with Coomassie Blue. Optimal induction conditions chosen (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig2">Fig. 2.</a> and <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig3">Fig. 3.</a>).</li></ol> | ||
+ | <a name="fig2"><img src="https://static.igem.org/mediawiki/2008/e/e7/July_31_st.jpg" width=350 /></a><var><b>Fig. 2.</b> Pellets from Z-omega induction with various IPTG concentrations<br> | ||
+ | The arrow shows place of our overexpressed protein:<br> | ||
+ | 1. 22°C 0.5 mM IPTG<br> | ||
+ | 2. 22°C 0.1 mM IPTG<br> | ||
+ | 3. 22°C 1 mM IPTG<br> | ||
+ | 4. Marker<br> | ||
+ | 5. negative control<br> | ||
+ | 6. 37°C 0.1 mM IPTG<br> | ||
+ | 7. 37°C 0.5 mM IPTG<br> | ||
+ | 8. 37°C 1 mM IPTG<br></var> | ||
+ | <a name="fig3"><img src="https://static.igem.org/mediawiki/2008/6/68/July_31_st_bis.jpg" width=350 /></a><var><b>Fig. 3. </b>Pellets and supernatants from Z-alpha induction with various IPTG concentrations<br> | ||
+ | The arrow shows place of our overexpressed protein:<br> | ||
+ | 1. Marker<br> | ||
+ | 2. pellet negative control<br> | ||
+ | 3. supernatant negative control<br> | ||
+ | 4. pellet 37°C 0.1 mM IPTG <br> | ||
+ | 5. supernatant 37°C 0.1 mM IPTG<br> | ||
+ | 6. sonicate 22°C 0.1 mM IPTG<br> | ||
+ | 7. sonicate 22°C 0.5 mM IPTG<br> | ||
+ | 8. pellet 37°C 0.5 mM IPTG<br> | ||
+ | 9. supernatant 37°C 0.5 mM IPTG<br></var> | ||
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+ | </html> | ||
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Latest revision as of 18:58, 26 October 2008
Cloning of truncated fragment of protein A (ΔA)Michał K.
Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alphaMichał K.
1-4. digested plasmids pACYC177 + OmpA-omega-ΔA-alpha 5. Marker Purification of proteins: Z-alpha and Z-omegaPiotr, Emilia
The arrow shows place of our overexpressed protein: 1. 22°C 0.5 mM IPTG 2. 22°C 0.1 mM IPTG 3. 22°C 1 mM IPTG 4. Marker 5. negative control 6. 37°C 0.1 mM IPTG 7. 37°C 0.5 mM IPTG 8. 37°C 1 mM IPTG Fig. 3. Pellets and supernatants from Z-alpha induction with various IPTG concentrations The arrow shows place of our overexpressed protein: 1. Marker 2. pellet negative control 3. supernatant negative control 4. pellet 37°C 0.1 mM IPTG 5. supernatant 37°C 0.1 mM IPTG 6. sonicate 22°C 0.1 mM IPTG 7. sonicate 22°C 0.5 mM IPTG 8. pellet 37°C 0.5 mM IPTG 9. supernatant 37°C 0.5 mM IPTG
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