Team:ETH Zurich/lab/protocols

From 2014.igem.org

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(PCR procol for phusion DNA polymerase)
(MakerBot 3D-printer)
 
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===NanoDrop 2000, UV-Vis Spectrophotometer===
===NanoDrop 2000, UV-Vis Spectrophotometer===
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[[File:ETH Zurich 2014_NanoDrop.jpg|float|300px|thumb|Our NanoDrop spectrophotometer]]  
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[[File:ETH Zurich 2014_NanoDrop.jpg|float|300px|thumb|'''Figure 1''' Our NanoDrop spectrophotometer]]  
A Thermo Scientific NanoDrop® 2000 spectrophotometer was used to determine the concentrations of our plasmids after miniprep.  
A Thermo Scientific NanoDrop® 2000 spectrophotometer was used to determine the concentrations of our plasmids after miniprep.  
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*Small sample measurement in NanoQuant Plate
*Small sample measurement in NanoQuant Plate
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[[File:ETH Zurich 2014 Tecan.jpg|center|500px|thumb|Tecan Infinite M200 Pro™]]  
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[[File:ETH Zurich 2014 Tecan.jpg|center|500px|thumb|'''Figure 2'''Tecan Infinite M200 Pro™]]  
Please visit the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan webpage] for more information.
Please visit the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan webpage] for more information.
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<br>
Please visit the [http://www.heraco.se/images/user/PDF/Novaspec%20Plus.pdf Novaspec™ Plus Visible Spectrophotometer webpage] for more information.
Please visit the [http://www.heraco.se/images/user/PDF/Novaspec%20Plus.pdf Novaspec™ Plus Visible Spectrophotometer webpage] for more information.
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[[File:ETH Zurich 2014_Novaspec.jpg|center|400px|thumb|Novaspec Plus™ Visible Spectrophotometer]]
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[[File:ETH Zurich 2014_Novaspec.jpg|center|400px|thumb|'''Figure 3''' Novaspec Plus™ Visible Spectrophotometer]]
===Biostep Dark-Hood DH-50™  and the Argus-X1™ software===
===Biostep Dark-Hood DH-50™  and the Argus-X1™ software===
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Please visit the [http://www.biostep.de/i18n_en/products/Bio_Imaging_components_1776/Tripod__dark_hoods_and_accessories_1863/Dark_hoods_1865/Dark_Hood_DH_50_6/index.html Biostep Dark-Hood DH-50™ webpage] for more information.
Please visit the [http://www.biostep.de/i18n_en/products/Bio_Imaging_components_1776/Tripod__dark_hoods_and_accessories_1863/Dark_hoods_1865/Dark_Hood_DH_50_6/index.html Biostep Dark-Hood DH-50™ webpage] for more information.
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[[File:ETH Zurich 2014_Biostep Dark-Hood DH-50™.jpg|center|300px|thumb|Biostep Dark-Hood DH-50™]]
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[[File:ETH Zurich 2014_Biostep Dark-Hood DH-50™.jpg|center|300px|thumb|'''Figure 4''' Biostep Dark-Hood DH-50™]]
===Biostep argusX1™ basic licence===
===Biostep argusX1™ basic licence===
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Please visit the [http://www.eppendorf.com/int/index.php?pb=5ffa644d89305ae7&action=products&contentid=1&catalognode=10019&productpage=1 Eppendorf Centrifuge 5810 R webpage] for more information.
Please visit the [http://www.eppendorf.com/int/index.php?pb=5ffa644d89305ae7&action=products&contentid=1&catalognode=10019&productpage=1 Eppendorf Centrifuge 5810 R webpage] for more information.
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[[File:ETH Zurich 2014_Eppendorf Centrifuge 5810 R.jpg|center|300px|thumb|Eppendorf Centrifuge 5810 R]]
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[[File:ETH Zurich 2014_Eppendorf Centrifuge 5810 R.jpg|center|300px|thumb|'''Figure 5''' Eppendorf Centrifuge 5810 R]]
===Eppendorf Centrifuge 5424 R===
===Eppendorf Centrifuge 5424 R===
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Please visit the [http://www.eppendorf.com/int/index.php?sitemap=2.1&pb=92b8536165b27ad1&action=products&contentid=1&catalognode=22420&productpage=1 Eppendorf Centrifuge 5424 R webpage] for more information.
Please visit the [http://www.eppendorf.com/int/index.php?sitemap=2.1&pb=92b8536165b27ad1&action=products&contentid=1&catalognode=22420&productpage=1 Eppendorf Centrifuge 5424 R webpage] for more information.
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[[File:ETH Zurich 2014 Eppendorf Centrifuge 5424 R.jpg|center|300px|thumb|Eppendorf Centrifuge 5424 R]]
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[[File:ETH Zurich 2014 Eppendorf Centrifuge 5424 R.jpg|center|300px|thumb|'''Figure 6''' Eppendorf Centrifuge 5424 R]]
===Harvard Pump 11 Plus===
===Harvard Pump 11 Plus===
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Please visit the [https://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_44010_-1_HAI_ProductDetail___ Harvard Pump 11 Plus webpage] for more information.
Please visit the [https://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_44010_-1_HAI_ProductDetail___ Harvard Pump 11 Plus webpage] for more information.
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[[File:ETH Zurich 2014_Harvard Apparatus syringe Pump 11 Plus.jpg|center|400px|thumb|Harvard Apparatus syringe Pump 11 Plus]]
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[[File:ETH Zurich 2014_Harvard Apparatus syringe Pump 11 Plus.jpg|center|400px|thumb|'''Figure 7''' Harvard Apparatus syringe Pump 11 Plus]]
===BD LSRFortessa™ Flow Cytometer System===
===BD LSRFortessa™ Flow Cytometer System===
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Please visit the [http://www.bdbiosciences.com/documents/bd_lsrfortessa_brochure.pdf BD LSRFortessa™ Flow Cytometer System webpage] for more information.
Please visit the [http://www.bdbiosciences.com/documents/bd_lsrfortessa_brochure.pdf BD LSRFortessa™ Flow Cytometer System webpage] for more information.
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[[File:ETH Zurich 2014_BD LSRFortessa™ Flow Cytometer System.jpg|center|400px|thumb|BD LSRFortessa™ Flow Cytometer System]]
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[[File:ETH Zurich 2014_BD LSRFortessa™ Flow Cytometer System.jpg|center|400px|thumb|'''Figure 8''' BD LSRFortessa™ Flow Cytometer System]]
===FACSDiva™ software===
===FACSDiva™ software===
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The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot with MakerWare software; MakerBotIndustries, Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene (ABS, about US$160 per kg). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively.
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot with MakerWare software; MakerBotIndustries, Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene (ABS, about US$160 per kg). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively.
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[[File:ETH Zurich MakerBot.jpg|center|400px|thumb|2nd generation MakerBot]]
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[[File:ETH Zurich MakerBot.jpg|center|400px|thumb|'''Figure 9''' 2nd generation MakerBot]]
<html></article></html>
<html></article></html>
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===Defined M9 medium with 0.4 % glycerol===
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Suspend/dissolve the sterile compounds in purified water to give 1 L final volume:
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{| border="0"
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|-
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| align="left"  width="300"| [http://openwetware.org/wiki/M9_salts M9 salts, 5x] || align="left" | 200 mL
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|-
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| align="left" | 30% (v/v) Glycerol || align="left" | 13.33 mL
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|-
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| align="left" | 10% (w/v) CAA || align="left" | 100 mL
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|-
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| align="left" | Thiamin (10 g/L) || align="left" |  2 mL
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|-
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| align="left" | TE-US solution<sup>[[Team:ETH_Zurich/project/references#refPanke|[33]]]</sup> || align="left" | 1 mL
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|-
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| align="left" | 1M CaCl<sub>2</sub> || align="left" | 0.5 mL
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|-
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| align="left" | 1M MgSO<sub>4</sub> || align="left" | 2 mL
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|}
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<br/>
===SOC===
===SOC===
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===Preparation of chemically competent ''E. coli''===
===Preparation of chemically competent ''E. coli''===
   
   
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Method
Method
#Design mutagenesis primer(s). The targeted mutation should be in the middle of the primer. Design your primers (including the mutations) to have a Tm >=78°C.
#Design mutagenesis primer(s). The targeted mutation should be in the middle of the primer. Design your primers (including the mutations) to have a Tm >=78°C.
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#Purify template plasmid from a dam+ E. coli strain via miniprep.
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#Purify template plasmid from a dam+ ''E. coli'' strain via miniprep.
#Set up mutagenesis PCR mix:
#Set up mutagenesis PCR mix:
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Latest revision as of 23:45, 17 October 2014

iGEM ETH Zurich 2014