Difference between revisions of "Team:Goettingen/Experiments"

Line 208: Line 208:
 
<p>&nbsp;</p>
 
<p>&nbsp;</p>
 
<p><span style="text-decoration: underline;">A: Stock reagents</span></p>
 
<p><span style="text-decoration: underline;">A: Stock reagents</span></p>
<p>1M IPTG (4,76 g in 20ml Millipore-H2O) filter with blue 0,22 um sterile filter and freeze at -20˚C</p>
+
<p>1M IPTG (4,76 g in 20ml Millipore-H2O) filter with blue 0,22 µm sterile filter and freeze at -20˚C</p>
<p>50mg/ml Kanamycin in Millipore H2O, filter with blue 0,22 um sterile filter and freeze at -20˚C</p>
+
<p>50mg/ml Kanamycin in Millipore H2O, filter with blue 0,22 µm sterile filter and freeze at -20˚C</p>
<p>100mg/ml Ampicillin in 50% ethanol, filter with blue 0,22 um sterile filter and freeze at -20˚C</p>
+
<p>100mg/ml Ampicillin in 50% ethanol, filter with blue 0,22 µm sterile filter and freeze at -20˚C</p>
<p>25mg/ml BCIP (in DMF) filter with blue 0,22 um sterile filter and keep in a falcon tube wrapped with aluminum foil at 4˚C. BCIP: Biomol Nr. 2291 (MG 433,64)</p>
+
<p>25mg/ml BCIP (in DMF) filter with blue 0,22 µm sterile filter and keep in a falcon tube wrapped with aluminum foil at 4˚C. BCIP: Biomol Nr. 2291 (MG 433,64)</p>
 
<p>Glycerol (99%), autoclaved and kept at room temperature</p>
 
<p>Glycerol (99%), autoclaved and kept at room temperature</p>
 
<p>&nbsp;</p>
 
<p>&nbsp;</p>

Revision as of 17:00, 17 September 2015



Media/Buffer

LB Medium

"Fat" LB Medium

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

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

Bradford Assay

Activity Screens

Esterase activity test

Phosphatase 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

Counting iGEM Goettingen2015.jpeg