Team:Warsaw/Calendar-Main/3 September 2008

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(New page: {{WarNotebook}} <!-- do not edit above me! --> Oczyszczanie białek: A-alfa, Z-alfa i Z-omega Piotr zawieszenie próbek w PBS, sonikacja, zwirowanie, do supernatantu i osadu osobn...)
 
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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>
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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 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 was stained with Coomassie Blue. Optimal induction conditions chosen.</li>
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<li>A-alpha in Rosetta strain was cultured overnight.</li></ol></p>
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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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The arrow shows place of our overexpressed protein:<br>
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1.  22&deg;C 0.5 mM IPTG 5 h<br>
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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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Oczyszczanie białek: A-alfa, Z-alfa i Z-omega
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Piotr
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zawieszenie próbek w PBS, sonikacja, zwirowanie, do supernatantu i osadu osobno dodanie buforu lizującego (opisać skład gdzieś) gotować przez 10 minut, na żel poliakryl 12% (zrobić protokół) nałożyć  ilość zlizowanych bakterii odpowiadającą 100mikrolitrom chodowli o OD=1, elektroforeza (zrobić protokół), barwienie i odbarwianie qmasim (zrobić protokuł)
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wybranie optymalnych warunków
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zaszczepienie A-alfa, Z-alfa i Z-omega
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Latest revision as of 21:04, 26 October 2008

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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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