Team:Marburg/Notebook:October

From 2013.igem.org

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<span class="aim-desc">Test of disruption method: Glass beads.
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<span class="aim-desc">Test of glass beads disruption method.
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     <legend><a name="soni">Cell disruption using ball mill</a></legend>
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     <legend><a name="soni">Cell disruption by means of ball mill</a></legend>
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     <div class="aim">
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<span class="aim">Aim:</span>
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<span class="aim-desc">Test of disruption method: ball mill.
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<span class="aim-desc">Test of ball mill disruption method.
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<li>Two steel balls were added &rarr; Tube was shock freezed again.</li>
<li>Two steel balls were added &rarr; Tube was shock freezed again.</li>
<li>Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.</li>
<li>Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.</li>
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</div>
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</fieldset>
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<!-- Cell disruption -->
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<fieldset class="experiment sonification">
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    <legend><a name="soni">Cell disruption by means of sonification</a></legend>
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    <div class="investigator">
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<span class="inv">Investigator:</span>
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<span class="inv-names">Franzi</span>
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    <div class="aim">
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<span class="aim">Aim:</span>
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<span class="aim-desc">Test of sonification disruption method.
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        </span>
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</div>
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<div class="exp-content">
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<p><ul class="digest">
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<li>4 ml cell culture was harvested (5 min centrifugation at full speed.</li>
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<li>The Supernatent was discarded and the pellet frozen in liquid nitrogen.</li>
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<li>The frozen pellet was resuspended in 0.68 ml IP buffer with 3.4 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high).</li>
 +
<li>Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.</li>
 +
<li>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice.</li>
 +
<li>The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone.</li>
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<li>The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</li>
</ul>
</ul>
</div>
</div>
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<div class="exp-content">
<div class="exp-content">
<p>10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.</p>
<p>10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.</p>
-
<p>Frozen cell-pellets were resuspended in 1,7 ml IP-buffer with 8,5 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.</p>
+
<p>Frozen cell-pellets were resuspended in 1,7 ml IP buffer with 8,5 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.</p>
<p>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</p>
<p>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</p>
<p>10 µg of each sample were used for SDS-PAGE (Westernblot).</p>
<p>10 µg of each sample were used for SDS-PAGE (Westernblot).</p>

Revision as of 09:37, 28 October 2013

Notebook: October Next Previous

19.10.2013

Light-inducible promoter
Investigators: Domenica and Dominik
Aim: Characterization of the light-inducible promotor PfcpB (BBa_K1071003).

Erlenmeyer flask with 50 mL P. tricornutum culture was coated with aluminium foil to prevent induction of PfcpB.

21.10.2013

Cell disruption by means of glass beads
Investigator: Dominik
Aim: Test of glass beads disruption method.

  • 4 ml cell culture was harvested.
  • Pellet was dissolved in 1 mL PBS.
  • 0.5 ml glass beads were added.
  • Tubes were shaken for 3 min → color shift from brown to green.
  • TCA precipitation was performed.
Cell disruption by means of ball mill
Investigator: Domenica
Aim: Test of ball mill disruption method.

  • 4 ml cell culture was harvested.
  • Dilution in 300 µL 8M urea buffer.
  • Shock freezing of suspension by means of liquid nitrogen.
  • Two steel balls were added → Tube was shock freezed again.
  • Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.
Cell disruption by means of sonification
Investigator: Franzi
Aim: Test of sonification disruption method.

  • 4 ml cell culture was harvested (5 min centrifugation at full speed.
  • The Supernatent was discarded and the pellet frozen in liquid nitrogen.
  • The frozen pellet was resuspended in 0.68 ml IP buffer with 3.4 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high).
  • Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.
  • 1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice.
  • The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone.
  • The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).

23.10.2013

Sonification
Investigators: Franzi, Marco
Aim: Quantification of the light-inducible promoter PfcpB (BBa_K1071003)
→ Cell disruption, protein precipitation and SDS-PAGE.

10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.

Frozen cell-pellets were resuspended in 1,7 ml IP buffer with 8,5 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.

1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).

10 µg of each sample were used for SDS-PAGE (Westernblot).

Amido black assay
Investigators: Franzi, Dominik, Domenica
Aim: Quantification of PfcpB → Quantification of protein amount.

5 µl of protein (in ureabuffer) were used.

    Measured protein concentrations:
  • Dark: 2,10 µg/µl
  • Red: 2,49 µg/µl
  • Green: 2,80 µg/µl
  • Blue: 3,23 µg/µl