Team:Marburg/Notebook:October

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<a name="23-10-2013">23.10.2013</a>
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    <legend><a name="soni">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, Marco</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">Quantification of P<sub>fcpB</sub> &rarr; Cell disruption, protein precipitation and SDS-PAGE.</span>
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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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<fieldset class="experiment amidoblack">
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    <legend><a name="amido">Amido black assay</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, Dominik, Domenica</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">Quantification of P<sub>fcpB</sub> &rarr; Quantification of protein amount.</span>
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</div>
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<div class="exp-content">
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<p>5 µl of protein (in ureabuffer) were used.</p
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<ul class="amido"> Measured protein concentrations:
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<li>Dark: 2,10 µg/µl</li>
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<li>Red: 2,49 µg/µl</li>
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<li>Green: 2,80 µg/µl</li>
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<li>Blue: 3,23 µg/µl</li>
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Revision as of 13:06, 27 October 2013

Notebook: October Next Previous

23.10.2013

Sonification
Investigator: Franzi, Marco
Aim: Quantification of PfcpB → 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

    • Retrieved from "http://2013.igem.org/Team:Marburg/Notebook:October"