Team:Warsaw/Calendar-Main/3 September 2008

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<h3>Purification of proteins: A-alpha, Z-alpha and Z-omega<br>Piotr</h3>
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<h3>Purification of proteins: A-alpha</h3><h4>Piotr, Emilia</h4>
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<p><ol><li>Samples were resuspended in PBS, sonicated and centrifuged.</li>  
<p><ol><li>Samples were resuspended in PBS, sonicated and centrifuged.</li>  
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<li>Lysis buffer were added separately to pellet and supernatant, then samples were boiled for 10 minutes. </li>
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<li>Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min. </li>
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<li>Lysates loaded on 12% poliacrylamide gel (amount relating to 100 ul of OD=1,0 culture)</li>
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<li>Lysates were loaded on 12% polyacrylamide gel (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/3_September_2008#fig1">Fig. 1.</a>) (amount relating to 100 μl of OD=1.0 culture).</li>
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<li>Gel stained with Coomassie Blue. Optimal induction conditions chosen</li>
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<li>Gel was stained with Coomassie Blue. Optimal induction conditions chosen.</li>
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<li>A-alpha, Z-alpha and Z-omega inoculated in the overnight culture (Rosetta strain)</li></ol></p>
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<li>A-alpha in Rosetta strain was cultured overnight.</li></ol></p>
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<br><br>
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<h3>Protein A mutagenesis<br>Paweł</h3>
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<p><ol><li>PNK phosporylation of mutagenesis for 30 minutes at 37C.</li>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/b/bd/September_3_rd.jpg" width=300 /></a><var><b>Fig. 1.</b> Supernatants from A-alpha induction with various IPTG concentrations<br>
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<li>PNK heat-inactivated by incubation at 75C for 15 minutes.</li>
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The arrow shows place of our overexpressed protein:<br>  
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<li>T4 ligase added and incubated for 1h at room temperature.</li>
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1. 22&deg;C 0.5 mM IPTG 5 h<br>
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<li>T4 ligase inactivated by incubation at 55C for 20 min, then 5U of DpnI added and incubated at 37C for 3 hours.</li> <li>After DpnI treatment mutagenesis transformed into TOP10 and plated on LB+kan plates.</li></ol></p>
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2.  Marker<br>
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3. 22&deg;C 0.5 mM IPTG overnight<br>
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4.  22&deg;C 1 mM IPTG 5 h<br>
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5. 22&deg;C 1 mM IPTG overnight<br>
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6.  37&deg;C 0.5 mM IPTG 5 h<br>
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7. 37&deg;C 0.5 mM IPTG overnight<br>
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8.  37&deg;C 1 mM IPTG 5 h<br>
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9.  37&deg;C 1 mM IPTG overnight<br>
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10. Marker<br></var>
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Purification of proteins: A-alpha

Piotr, Emilia

  1. Samples were resuspended in PBS, sonicated and centrifuged.
  2. Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min.
  3. Lysates were loaded on 12% polyacrylamide gel (Fig. 1.) (amount relating to 100 μl of OD=1.0 culture).
  4. Gel was stained with Coomassie Blue. Optimal induction conditions chosen.
  5. A-alpha in Rosetta strain was cultured overnight.

Fig. 1. Supernatants from A-alpha induction with various IPTG concentrations
The arrow shows place of our overexpressed protein:
1. 22°C 0.5 mM IPTG 5 h
2. Marker
3. 22°C 0.5 mM IPTG overnight
4. 22°C 1 mM IPTG 5 h
5. 22°C 1 mM IPTG overnight
6. 37°C 0.5 mM IPTG 5 h
7. 37°C 0.5 mM IPTG overnight
8. 37°C 1 mM IPTG 5 h
9. 37°C 1 mM IPTG overnight
10. Marker


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