Team:LMU-Munich/Lab Notebook/Protocols

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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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===Protocols for the work with ''B. subtilis''===
===Protocols for the work with ''B. subtilis''===
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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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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'']
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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Isolation of pure genomic DNA from ''B. subtilis''.
Isolation of pure genomic DNA from ''B. subtilis''.
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6. [https://static.igem.org/mediawiki/2012/7/7a/LMU-Munich_2012_Long-Flanking_Homology_PCR.pdf Long Flanking Homology PCR]
6. [https://static.igem.org/mediawiki/2012/7/7a/LMU-Munich_2012_Long-Flanking_Homology_PCR.pdf Long Flanking Homology PCR]
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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7. [https://static.igem.org/mediawiki/2012/6/6d/LMU-Munich_2012_Clean_deletions_in_Bacillus_subtilis.pdf Clean deletion of genomic fragments in ''Bacillus subtilis'']
7. [https://static.igem.org/mediawiki/2012/6/6d/LMU-Munich_2012_Clean_deletions_in_Bacillus_subtilis.pdf Clean deletion of genomic fragments in ''Bacillus subtilis'']
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Protocol for the production of markerless gene deletions. You will need the [http://www.ncbi.nlm.nih.gov/pubmed/15528558 pMAD] plasmid. This method is more time consuming than inserting resistance cassettes but offers better cell perfomance.
Protocol for the production of markerless gene deletions. You will need the [http://www.ncbi.nlm.nih.gov/pubmed/15528558 pMAD] plasmid. This method is more time consuming than inserting resistance cassettes but offers better cell perfomance.
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8. [https://static.igem.org/mediawiki/2012/6/66/LMU-Munich_2012_Spore_germination_assay.pdf Spore germination assay]
8. [https://static.igem.org/mediawiki/2012/6/66/LMU-Munich_2012_Spore_germination_assay.pdf Spore germination assay]
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Protocol to count efficiency of sporulation and germination.
Protocol to count efficiency of sporulation and germination.
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9. [https://static.igem.org/mediawiki/2012/7/70/LMU-Munich_2012_%C3%9F-Galactosidase_Assay_B._subtilis.pdf ß-Galactosidase Assay ''B. subtilis'']
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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 how to measure ''lacZ'' gene activity in ''B. subtilis''
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Protocol to enrich the spore numbers and purification from vegetative cells.
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10. [https://static.igem.org/mediawiki/2012/7/70/LMU-Munich_2012_%C3%9F-Galactosidase_Assay_B._subtilis.pdf ß-Galactosidase Assay ''B. subtilis'']
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Protocol how to measure ''lacZ'' gene activity in ''B. subtilis''
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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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===Other protocols we used===
===Other protocols we used===
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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 dreck-prep
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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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Kompetente E.colis
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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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Ligation, Verdau
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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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E.coli trafo
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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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lacZ assay in E.coli
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Quantitative ''lacZ''-assay in ''E.coli''.
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Plate reader assay
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Mikroskopie der Sporen
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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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