Team:LMU-Munich/Lab Notebook/Protocols

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==Protocols==
 
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On this page, we will upload unique protocols to share with the public.
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On this page, we offer our unique protocols to the public.
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Since we work with ''Bacillus subtilis'' as chassis we focus on protocols how to work with that organism.
 
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'''Because ''Bacillus subtilis'' is our choice organism, we have focused on protocols specifically for ''Bacillus''.'''
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* [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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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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* [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/2012/6/66/LMU-Munich_2012_Spore_germination_assay.pdf Spore germination assay]
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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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* [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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'''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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* [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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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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* [https://static.igem.org/mediawiki/2012/7/7a/LMU-Munich_2012_Long-Flanking_Homology_PCR.pdf Long Flanking Homology PCR]
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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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* [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 for the transformation of ''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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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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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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</div>
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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....)]
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Isolation of pure genomic DNA from ''B. subtilis''.
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</div>
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<div class="box">
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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]
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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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</div>
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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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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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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.
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</div>
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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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</div>
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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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</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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<div class="box" style= "background-color:#e4f1d7">
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===Other protocols we used===
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</div>
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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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</div>
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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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</div>
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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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</div>
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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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</div>
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Latest revision as of 02:00, 27 September 2012

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