Team:Marburg/Notebook:October

From 2013.igem.org

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<a name="21-10-2013">21.10.2013</a>
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    <legend><a name="soni">Cell disruption by means of glass beads</a></legend>
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<span class="inv">Investigator:</span>
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<span class="inv-names">Dominik</span>
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<span class="aim">Aim:</span>
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<span class="aim-desc">Test of disruption method: Glass beads.
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<li>4 ml cell culture was harvested.</li>
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<li>Pellet was dissolved in 1 mL PBS.</li>
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<li>0.5 ml glass beads were added.</li>
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<li>Tubes were shaken for 3 min &rarr; color shift from brown to green.</li>
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<li>TCA precipitation was performed.</li>
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<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>
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<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>
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<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>
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<p>10 µg of each sample were used for SDS-PAGE (Westernblot).</p>
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     <legend><a name="soni">Sonification</a></legend>
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<span class="inv">Investigators:</span>
<span class="inv-names">Franzi, Marco</span>
<span class="inv-names">Franzi, Marco</span>
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Revision as of 21:45, 27 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 disruption method: Glass beads.

  • 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.

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).

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
Investigator: 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