Difference between revisions of "Team:Goettingen/Experiments"

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<p>For calculating protein concentrations of an unknown sample the traditional way is to use a standard curve that is generated from known protein standards. Readily available protein standards contain bovine serum albumin (BSA) or bovine gamma globulin (&gamma;-globulin, IgG). The calculated result is an estimation of protein concentration since Bradford protein assays do show significant protein-to-protein variation.</p>
 
<p>For calculating protein concentrations of an unknown sample the traditional way is to use a standard curve that is generated from known protein standards. Readily available protein standards contain bovine serum albumin (BSA) or bovine gamma globulin (&gamma;-globulin, IgG). The calculated result is an estimation of protein concentration since Bradford protein assays do show significant protein-to-protein variation.</p>
 
<p>For our Bradford assay we got an already prepared standard solution with a factor of 15.924.</p><p>Caution: Store the Bradford solution in the dark, it is light sensitive.</p>
 
<p>For our Bradford assay we got an already prepared standard solution with a factor of 15.924.</p><p>Caution: Store the Bradford solution in the dark, it is light sensitive.</p>
 +
<p>&nbsp;</p>
 
<p><strong>Sample preparation:</strong></p>
 
<p><strong>Sample preparation:</strong></p>
 
<p>Measurement takes place against 20 &mu;L water (blank).</p>
 
<p>Measurement takes place against 20 &mu;L water (blank).</p>
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     <li>Immediately measure the samples at 595 nm</li>
 
     <li>Immediately measure the samples at 595 nm</li>
 
</ul>
 
</ul>
 +
<p>&nbsp;</p>
 
<p>Calculation of protein concentration:</p>
 
<p>Calculation of protein concentration:</p>
 
<p><br /> &nbsp;</p>
 
<p><br /> &nbsp;</p>

Revision as of 19:24, 16 September 2015



Workflow.jpeg

Media

LB Medium

"Fat" LB Medium

Phosphatase Activity plates, Sperber media

Esterase Activity plates, with 1% Tributyrin

Cellulase activity plates

Cloning Methods

PCR product purification using QIAquick® PCR Purification Kit (QIAGEN)

PCR Gel extraction, peqGOLD Gel Extraction Kit

Blunt End Ligation in pJET1.2 vector –Clone JET PCR Cloning Kit–Thermo Scientific

TOPO® Cloning protocol usingChampion™ pET Directional TOPO® Expression Kits. Thermo Fisher Scientific

Plasmid transformation into chemically competent E. coli

Electroporation of BL21 cells with pJET_RFP

Plasmid Extraction - using QIAprep Spin Miniprep Kit (QIAGEN)

Plasmid Extraction - using peqGOLD Plasmid Miniprep Kit I (PEQLAB Technologies)

Competent Cells

Preparation of competent E.coli cells

Transformation Efficiency Kit, RFP construct (iGEM)

Protein Extraction and Purification

Protein Extraction (French Press) and Purification (Protino® Ni-IDA 2000 His-Tag protein purification, Macherey-Nagel)

Bradford Assay

Activity Screens

Esterase activity test

Cellulase activity screening

Restriction Controls

Aan I (Psi I ) - thermo fisher scientific - restriction control protocol

Double digestion restriction control

Restriction control using fast and slow digestion enzymes

Scafoldin Restriction control

Esterase Restriction Control

Phosphatase Restriction Control

PCR Preparation Methods

Colony PCR

Phusion PCR

Sequencing

Protocol for Sanger sequencing

Overnight Sanger Sequencing

Fluorescence Microscopy

RFP microscopy