Difference between revisions of "Team:Goettingen/Experiments"

 
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}
 
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#menu1, #menu2, #menu3, #menu4, #menu5, #menu6, #menu7, #menu8, #menu9, #menu10, #menu11, #menu12, #menu13, #menu14, #menu15, #menu16, #menu17, #menu18, #menu19, #menu20, #menu21, #menu22, #menu23, #menu24, #menu25, #menu26, #menu27, #menu28, #menu29, #menu30, #menu31, #menu32, #menu33, #menu34, #menu35, #menu36, #menu37, #menu38, #menu39, #menu40, #menu41{
 
     display:none;
 
     display:none;
 
     width:90%;
 
     width:90%;
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</div>
 
</div>
<h2> Activity Screens </h2>
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<h2> Activity Screens & Tests</h2>
 
                     <a href="" onClick=" $('#menu9').slideToggle(400, function callback() {  }); return false;"><h1>Esterase activity test</h1></a>
 
                     <a href="" onClick=" $('#menu9').slideToggle(400, function callback() {  }); return false;"><h1>Esterase activity test</h1></a>
 
<div id="menu9">
 
<div id="menu9">
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     <li>Incubate plates at 37<sup>o</sup>C for minimum 3-4 days (timing can be extended depending on bacterial strain).</li>
 
     <li>Incubate plates at 37<sup>o</sup>C for minimum 3-4 days (timing can be extended depending on bacterial strain).</li>
 
     <li>After 3-4 days look for plates having clear zone of activity around cellulose substrate.</li>
 
     <li>After 3-4 days look for plates having clear zone of activity around cellulose substrate.</li>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu41').slideToggle(300, function callback() {  }); return false;"><h1 style="color:white;"> Enzymatic Activity Test for Cellulase </h1></a>
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<div id="menu41">
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 +
 +
<p>
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    Solutions:
 +
</p>
 +
<p>
 +
    Buffer:
 +
</p>
 +
<p>
 +
    Tris (MW: 121.14 g/mol) 50 mM
 +
</p>
 +
<p>
 +
    dH<sub>2</sub>O ad 1 l
 +
</p>
 +
<p>
 +
    pH 7
 +
</p>
 +
<p>
 +
    Substrate solution:
 +
</p>
 +
<p>
 +
    Carboxymethil cellulose 1 % (w / v)
 +
</p>
 +
<p>
 +
    ddH<sub>2</sub>O ad 1 l
 +
</p>
 +
<p>
 +
    DNSA reagent solution:
 +
</p>
 +
<p>
 +
    Dinitrosalycylic acid 10 g
 +
</p>
 +
<p>
 +
    Phenol 2 ml
 +
</p>
 +
<p>
 +
    K Na Tartrate 200 g
 +
</p>
 +
<p>
 +
    NaOH 10 g
 +
</p>
 +
<p>
 +
    Na<sub>2</sub>SO<sub>3</sub> 0.5 g
 +
</p>
 +
<p>
 +
    dH<sub>2</sub>O ad 1 l
 +
</p>
 +
<p>
 +
    Store at 4°C, protected from light
 +
</p>
 +
<p>
 +
    Procedure:
 +
</p>
 +
<p>
 +
    250 µl substrate solution
 +
</p>
 +
<p>
 +
    100 µl Tris-Buffer
 +
</p>
 +
<p>
 +
    x µl enzyme solution
 +
</p>
 +
<p>
 +
    ad 500 µl ddH<sub>2</sub>O
 +
</p>
 +
<p>
 +
    Incubate setup as described above at 37°C for 20 min.
 +
</p>
 +
<p>
 +
    Add 750 µl DNSA. Heat the setup at 96°C for 15 min.
 +
</p>
 +
<p>
 +
    Put mixture on ice, centrifuge shortly at 4°C, 13000 x g.
 +
</p>
 +
<p>
 +
    Measure OD<sub>575</sub> against same setup without enzyme as a blank.
 +
</p>
 
</div>
 
</div>
  

Latest revision as of 00:00, 19 September 2015



Media/Buffer

LB Medium

"Fat" LB Medium

Media and Culture Methods for Dockerin Organisms of Origin

Phosphatase Activity plates, Sperber media

Esterase Activity plates, with 1% Tributyrin

Cellulase activity plates

1x TAE Buffer

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)

Sticky End T4 Ligation (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

Induction, harvest and disruption of expression cultures

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

Affinity chromatography of His-tagged proteins

Size Exclusion Chromatography

Concentration of protein solutions

Bradford Assay

SDS Polyacrylamid Gel Electrophoresis

Western Blot

Activity Screens & Tests

Esterase activity test

Phosphatase activity test

Cellulase activity screening

Enzymatic Activity Test for Cellulase

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

Counting iGEM Goettingen2015.jpeg