Team:Warsaw/Calendar-Main/31 July 2008
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+ | <h3> Cloning of truncated fragment of protein A</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 + deltaA</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + deltaA</a>). </li> | ||
+ | <li> Transformants plating on LB + kanamycin. </li></ol></p> | ||
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<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 (Fig.3.) </li></ol> | <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 (Fig.3.) </li></ol> | ||
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+ | <img src="https://static.igem.org/mediawiki/2008/2/2e/31_july.jpg" width=250/> <var><b>Fig. 3. Control SacI/BamHI digests of isolated plasmids</b><br> | ||
+ | 1-4. digested plasmids pACYC177 + OmpA-omega-deltaA-alpha<br> | ||
+ | 5. Marker<br></var> | ||
- | <h3> | + | <h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr, Emilia</h4> |
- | <h4> | + | <p><ol><li>Samples were resuspended in PBS, sonicated and centrifuged.</li> |
- | <p><ol> | + | <li>Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min. </li> |
- | <li> | + | <li>Lysates loaded on 12% poliacrylamide gel (amount relating to 100 μl of OD=1.0 culture).</li> |
- | <li> | + | <li>Gel stained with Coomassie Blue. Optimal induction conditions chosen (Fig. 1 and Fig. 2).</li></ol> |
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<img src="https://static.igem.org/mediawiki/2008/e/e7/July_31_st.jpg" width=350 /><var>Fig. 1. <b>Pellets from Z-omega induction with various IPTG concentrations<br> | <img src="https://static.igem.org/mediawiki/2008/e/e7/July_31_st.jpg" width=350 /><var>Fig. 1. <b>Pellets from Z-omega induction with various IPTG concentrations<br> | ||
The arrow shows place of our overexpressed protein:</b><br> | The arrow shows place of our overexpressed protein:</b><br> | ||
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9. supernatant 37°C 0.5 mM IPTG<br></var> | 9. supernatant 37°C 0.5 mM IPTG<br></var> | ||
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Revision as of 17:22, 26 October 2008
Cloning of truncated fragment of protein AMichał K.
Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alphaMichał K.
1-4. digested plasmids pACYC177 + OmpA-omega-deltaA-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. 2. 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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