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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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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This protocol gives the recipes for various media and the antibiotic concentrations used for ''B. subtilis''.
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However [[Team:LMU-Munich/Lab Notebook/Protocols#Other protocols we used|below]], you will also find other standard protocols we use.
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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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<div class="box" style= "background-color:#e4f1d7">
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===Protocols for the work with ''B. subtilis''===
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</div>
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Protocol for the transformation of ''B. subtilis''.
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<div class="box">
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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 basic growth- and storage conditions, and the recipes for various media and the antibiotic concentrations used for ''B. subtilis''.
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</div>
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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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<div class="box">
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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''.
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</div>
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<div class="box">
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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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</div>
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<div class="box">
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4. [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/vector_use ''Bacillus subtilis'' vectors]
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4. [https://static.igem.org/mediawiki/2012/6/6b/LMU-Munich_2012_Isolation_of_genomic_DNA_from_Bacillus_%28for_PCR....%29.pdf Isolation of chromosomal DNA from ''Bacillus subtilis'' (for PCR....)]
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How to work with integrative ''B. subtilis'' vectors and how to verify the integration.
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</div>
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<div class="box">
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5. [https://static.igem.org/mediawiki/2012/6/6b/LMU-Munich_2012_Isolation_of_genomic_DNA_from_Bacillus_%28for_PCR....%29.pdf Isolation of chromosomal DNA from ''Bacillus subtilis'' (for PCR....)]
Isolation of pure genomic DNA from ''B. subtilis''.
Isolation of pure genomic DNA from ''B. subtilis''.
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</div>
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<div class="box">
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5. [https://static.igem.org/mediawiki/2012/7/7a/LMU-Munich_2012_Long-Flanking_Homology_PCR.pdf Long Flanking Homology PCR]
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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]
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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6. [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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[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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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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An explanation of how we used this protocol for how we knocked out our germination genes using LFH.
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</div>
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<div class="box">
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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'']
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7. [https://static.igem.org/mediawiki/2012/6/66/LMU-Munich_2012_Spore_germination_assay.pdf Spore germination assay]
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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.
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</div>
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<div class="box">
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8. [https://static.igem.org/mediawiki/2012/6/66/LMU-Munich_2012_Spore_germination_assay.pdf Spore germination assay]
Protocol to count efficiency of sporulation and germination.
Protocol to count efficiency of sporulation and germination.
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</div>
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<div class="box">
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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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8. [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 to enrich the spore numbers and purification from vegetative cells.
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</div>
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<div class="box">
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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'']
Protocol how to measure ''lacZ'' gene activity in ''B. subtilis''
Protocol how to measure ''lacZ'' gene activity in ''B. subtilis''
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</div>
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<div class="box">
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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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</div>
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Other protocols we used:
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<div class="box" style= "background-color:#e4f1d7">
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===Other protocols we used===
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</div>
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E. coli + B. subtilis antibiotics concentrations
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<div class="box">
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[http://partsregistry.org/Help:Protocols/Competent_Cells Competent Cells]
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E.coli dreck-prep
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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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</div>
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Kompetente E.colis
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<div class="box">
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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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Ligation, Verdau
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Alkaline Lysis Plasmid Preparation for ''E. coli''
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</div>
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E.coli trafo
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<div class="box">
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[http://openwetware.org/wiki/Silver:_Restriction_Digest Restriction digest]
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How to test integration of B. subtilis vectors
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[http://openwetware.org/wiki/Silver:_Ligation Ligation]
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lacZ assay in E.coli
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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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</div>
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Plate reader assay
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<div class="box">
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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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Mikroskopie der Sporen
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Quantitative ''lacZ''-assay in ''E.coli''.
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</div>
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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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