Team:LMU-Munich/Lab Notebook/Protocols

From 2012.igem.org

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==Protocols==
 
On this page, we offer our unique protocols to the public.
On this page, we offer our unique protocols to the public.
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Because ''Bacillus subtilis'' is our choice organism, we have focused on protocols specifically for ''Bacillus''.
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'''Because ''Bacillus subtilis'' is our choice organism, we have focused on protocols specifically for ''Bacillus''.'''
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1. [https://static.igem.org/mediawiki/2012/5/56/LMU-Munich_2012_Media_for_Bacillus_subtilis.pdf Media and components for ''Bacillus subtilis'']
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'''1.''' [https://static.igem.org/mediawiki/2012/5/56/LMU-Munich_2012_Media_for_Bacillus_subtilis.pdf Growth, storage, media and components for ''Bacillus subtilis'']
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This protocol gives the recipes for various media and the antibiotic concentrations used for ''B. subtilis''.
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This protocol gives the basic growth- and storage conditions, and the recipes for various media and the antibiotic concentrations used for ''B. subtilis''.
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Growth conditions and storage
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'''2.''' [https://static.igem.org/mediawiki/2012/4/41/LMU-Munich_2012_Transformation_of_Bacillus_subtilis.pdf Transformation of ''Bacillus subtilis'']
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2. [https://static.igem.org/mediawiki/2012/4/41/LMU-Munich_2012_Transformation_of_Bacillus_subtilis.pdf Transformation of ''Bacillus subtilis'']
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Protocol for the transformation of ''B. subtilis''.
Protocol for the transformation of ''B. subtilis''.
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3. [https://static.igem.org/mediawiki/2012/0/00/LMU-Munich_2012_Isolation_of_chromosomal_DNA_from_Bacillus_subtilis_for_transformation.pdf Isolation of chromosomal DNA from ''Bacillus subtilis'' for transformation]
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'''3.''' [https://static.igem.org/mediawiki/2012/0/00/LMU-Munich_2012_Isolation_of_chromosomal_DNA_from_Bacillus_subtilis_for_transformation.pdf Isolation of chromosomal DNA from ''Bacillus subtilis'' for transformation]
A quick protocol for how to isolate genomic DNA from ''B. subtilis'' to transform it to a different strain. By this means, you can very easily combine different ''B. subtilis'' genotypes. Transformation of genomic DNA is much more efficient than plasmids. Isolated DNA with this protocol is not clean!
A quick protocol for how to isolate genomic DNA from ''B. subtilis'' to transform it to a different strain. By this means, you can very easily combine different ''B. subtilis'' genotypes. Transformation of genomic DNA is much more efficient than plasmids. Isolated DNA with this protocol is not clean!
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4. How to test integration of B. subtilis vectors
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4. [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/vector_use ''Bacillus subtilis'' vectors]
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How to work with integrative ''B. subtilis'' vectors and how to verify the integration.
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PCR protocol for long flanking homology PCR. You might need those PCR products for interrupting genes with resistance cassettes.
PCR protocol for long flanking homology PCR. You might need those PCR products for interrupting genes with resistance cassettes.
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[https://static.igem.org/mediawiki/2012/6/67/LMU-Munich_2012_Removal_of_germination_genes.pdf Example: Using LFH PCR to replace germination genes with resistance cassettes]
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An explanation of how we used this protocol for how we knocked out our germination genes using LFH.
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9. Mikroskopie der Sporen
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9. [https://static.igem.org/mediawiki/2012/e/e9/LMU-Munich_2012_Protocol_for_enhancement_of_mature_spore_numbers.pdf Enhancement of mature spore numbers]
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Protocol to enrich the spore numbers and purification from vegetative cells.
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11. Plate reader assay (can also be used with E. coli cells)
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11. [https://static.igem.org/mediawiki/2012/e/e6/LMU-Munich_2012_Protocol_Plate_Reader.pdf Luminescence measurement]
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This protocol explains how cultures were treated in advance of a plate reader assay.
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E. coli antibiotics concentrations
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[http://partsregistry.org/Help:Protocols/Competent_Cells Competent Cells]
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''E.coli'' competent cells and transformation. We used the [http://ecoliwiki.net/colipedia/index.php/XL-1_Blue XL1 blue strain].
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E.coli trafo
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[https://static.igem.org/mediawiki/2012/1/1f/LMU-Munich_2012_Alkaline_Lysis_Plasmid_Preparation.pdf Alkaline Lysis Plasmid Preparation]
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Alkaline Lysis Plasmid Preparation for ''E. coli''
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E.coli dreck-prep
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Ligation, Verdau
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[http://openwetware.org/wiki/Silver:_Restriction_Digest Restriction digest]
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[http://openwetware.org/wiki/Silver:_Ligation Ligation]
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Standard restriction digest and ligation. We often used longer incubation times (up to over night) and did not dephosphorylate the backbones, if they had incompatible sticky ends.
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Kompetente E.colis
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lacZ assay in E.coli
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[https://static.igem.org/mediawiki/2012/b/bb/LMU-Munich_2012_%C3%9F-Galactosidase_Assay_E._coli.pdf β-Galactosidase Assay in ''E. coli'']
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Quantitative ''lacZ''-assay in ''E.coli''.
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(Proteinase K)
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(Western Blot)
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Latest revision as of 02:00, 27 September 2012

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