<noautolink> <!-- Preferences start here * Set PAGETITLE = Barrick Lab :: Laboratory Protocols Preferences end here --> ---+!! Barrick Lab :: Laboratory Protocols *Table of Contents* %TOC% ---++ Creating Protocols * [[ProtocolHowTo][How to Create a Protocol]] <br> Best practices for designing a protocol and for putting it on the lab Wiki. * [[ProtocolUpdatesNeeded][Updates Needed]] <br> Request new protocols and changes to protocols. ---++ Lab Stocks and Practices * [[StandardMicrobiologicalPractices][Standard Microbiological Practices]] <br> aka the approximately Ten Commandments of microbiology lab research. * [[AutoclaveSterilization][Autoclave Sterilization]] <br> * [[GlasswareCleaning][Cleaning Glassware]] <br> * [[ProtocolsMediaRecipes][Media Recipes]] <br> Solid and liquid media for bacterial growth. * [[ProtocolsAntibioticStockSolutions][Antibiotics and Supplements]] <br> Stock solutions and working concentrations of antibiotics and media supplements. * [[ProtocolsReagentRecipes][Reagent and Buffer Recipes]] <br> How to prepare common stocks of chemical solutions used in lab. * [[ReferencePlasmids][Plasmids]] <br> Sequences and characteristics of plasmids commonly used in lab. * [[ProtocolsFreezingStrains][Freezing Strains]] <br> How to freeze and archive strain samples. * [[ProtocolsStrainDatabase][Strain Database]] <br> How to search and enter samples in the lab database. * [[ProtocolsReagentsPhusion][Phusion (pfu-sso7d) polymerase purification]] <br> Draft: How to prepare and harvest pfu-sso7d polymerase. <br> * [[ReferenceKeioASKACollection][Keio and ASKA Collections]] <br> Information about finding and using these _E. coli_ strains. * [[ProtocolsFindChemicalsWithSciFinder][Find chemicals using SciFinder]] <br> Search by structure, identify chemical synonyms, and find commercial sources. ---++ PCR and Sequencing * [[ProceduresPrimerDesign][Sequencing/Genotyping Primer Design]] <br> Design primers to validate mutations in evolved genomes. * _Escherichia coli_ REL606 Genome Resources: [[http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=413997&INPUT_SEQUENCE=NC_012967.1&log$=seqview_list_primer][Design Primers]] | [[http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&SET_SAVED_SEARCH=true&PAGE=MegaBlast&PROGRAM=blastn&GAPCOSTS=0%200&MATCH_SCORES=1,-2&DATABASE=nr&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&REPEATS=repeat_9606&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=m&EQ_MENU=Escherichia%20coli%20B%20str.%20REL606%20%28taxid%3A413997%29&SHOW_OVERVIEW=on&SHOW_LINKOUT=on&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=on&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&OLD_BLAST=false][Blast]] | [[http://www.ncbi.nlm.nih.gov/nuccore/254160123][Genbank]] | [[http://www.ncbi.nlm.nih.gov/nuccore/NC_012967.1?report=graph][Browser]] |[[http://www.ecocyc.org/ECOL413997/][BioCyc]] * _Acinetobacter baylyi_ ADP1 Genome Resources: [[http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&PAGE=MegaBlast&PROGRAM=blastn&GAPCOSTS=0%200&MATCH_SCORES=1,-2&BLAST_SPEC=MicrobialGenomes_62977&EQ_MENU=Acinetobacter%20sp.%20ADP1%20%28taxid%3A62977%29&DATABASE=Complete_Genomes&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=m&DB_GROUP=AllMG&SUBGROUP_NAME=All_genomes&DB_SUBGROUP=Complete_Genomes&SHOW_OVERVIEW=on&SHOW_LINKOUT=on&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=on&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&OLD_BLAST=false][BLAST]] | [[http://biocyc.org/organism-summary?object=ASP62977][BioCyc]] * Predicting transcription start sites in bacteria: [[http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb][BPROM]] | [[http://bioinformatics.biol.rug.nl/websoftware/ppp/ppp_start.php][PPP]] | [[http://www.fruitfly.org/seq_tools/promoter.html][NNPP]] * [[ProtocolsDegenerateBases][Notes about degenerate bases from IDT]] * [[ProtocolsOrderingPrimers][Ordering Primers]] | [[http://www.idtdna.com/ICMBSupplyCenter/Login.aspx][%ICON{external}% ICMB's IDT page]] * [[ProceduresPrimerDatabase][Primer Database]] * [[ProtocolsResuspendingPrimers][Resuspending Primers]] * [[ProtocolsStandardPCR][Standard PCR]] * [[ProtocolsTaq][Which polymerase is right for me?]] * [[ProtocolsDpnIDigestion][Easy DpnI Digestion]] * [[ProceduresOverlapPCR][Overlap PCR]] * [[ProceduresStandardAgaroseGel][Agarose Gel Electrophoresis]] * [[DNAConcentrationDetermination][DNA Concentration Determination]]<br>Protocols for Qubit, Nanodrop, and Agarose Gel DNA concentrations, and example data highlighting caveats for each. * [[ProcedureDNASequencing][DNA Sequencing]] * [[Protocols16SSequencing][16S rRNA Sequencing]]<br>16S rRNA PCR and sequencing to identify unknown microbes * Tools: [[http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi][Blast2Seq (NCBI)]] | [[http://www.ncbi.nlm.nih.gov/gorf/gorf.html][ORF Finder (NCBI)]] | [[http://www.ebi.ac.uk/Tools/emboss/transeq/index.html][Translate (EMBOSS)]] * [[MEGAWHOP][MEGAWHOP]] * [[IlluminaSequencing][Illumina Next Generation Sequencing]]<br> Protocol for NGS library generation ---++ Genetic Modification and Cloning * [[ProtocolsUVLibrary][UV Mutagenesis to create a mutation library]] <br> Make a library of mutants with UV irradiation. * [[ProtocolsAraMarker][Selecting Arabinose Marker Revertants]]<br>Direct selection of Ara+ revertants from Ara– strains. * [[ProtocolsElectrocompetentCells][Electrocompetent Cells]]<br>Make electrocompetent cell stocks for transformation. * [[ProtocolsEthanolPrecipitation][Ethanol Precipitation]]<br>Precipitate DNA to remove salts before electroporation. * [[ProtocolsRestrictionEnzymeCloning][Restriction Enzyme Cloning]]<br> Insert a target DNA sequence into a plasmid via restriction digest and ligation. * [[ProtocolsGeneGorgingMarker][Gene Gorging Neutral Markers]]<br>λ red genetic modification for mutations with a selectable or screenable marker using donor plasmid. For example, converting Ara+ strains to Ara–. * [[ProceduresGeneGorgingEvolvedAlleles][Gene Gorging Evolved Alleles]] <br>λ red genetic modification without a selectable or screenable phenotype using donor plasmid. * [[ProcedureFLPFRTRecombination][FLP-FRT Recombination]] <br>Induce targeted recombination (commonly used to eliminate the Kan<sup>R</sup> cassette from Keio strains). * [[ProcedureGenomeModificationDatsenkoWanner][Genome Modification (Datsenko and Wanner Method)]] <br>λ red genetic modification using a PCR product containing a selectable marker. * [[ProcedureGeneReplacements][Gene Replacements (Church Method)]]<br>Allelic exchange using pKOV integration. Generate unmarked mutations. * [[ProtocolsGibsonCloning][Gibson Cloning]]<br>Make complex constructs from multiple genetic parts. Master mix recipe, procedure, and Gibson walkthrough included. * [[ProtocolsSsdnaRecombination][lambda red mediated ssDNA gene modification]]<br>λ red mediated ssDNA oligo mutation reconstruction. * [[ProtocolsPsltsEditing][pSLTS genomic editing for E. Coli (Copley lab)]]<br>Scarless genome modification using pSLTS plasmids developed by Copley lab. * [[ProtocolsAcinetobacterTransformation][Transforming Acinetobacter baylyi ADP1]]<br>How to transform Acinetobacter baylyi ADP1 with plasmids or PCR products. * [[ProtocolsAcinetobacterGenomeManipulation][Acinetobacter baylyi ADP1 genome manipulation]]<br>How to make deletions, insertions, and other modifications to the Acinetobacter baylyi genome. * [[ProtocolsMegaprimerMegawhop][Megaprimer Megawhop Mutagenesis]] <br> Simple and reliable method for introducing specific mutations into plasmids. ---++ Mutation Rates * [[ProtocolsFluctuationTests][Fluctuation Tests]]<br>Measure bacterial mutation rates to antibiotic resistance, phage resistance, or reversion of auxotrophy. * [[ProceduresPhageFarming][Phage Stocks]]<br>Phage Lysate Preparation (T4,T5,T6) and Determining Phage Titer ---++ Genome Resequencing and Mutation Validation * [[ProceduresGenomicDNAResequencing][Genomic DNA Isolation]]<br>Isolate gDNA for genome resequencing. * [[ProceduresTargetedSequencing][Targeted Sequencing of a Specific Gene]]<br>To find mutations or validate predictions from whole-genome re-sequencing data. * [[ProceduresCalculatingMutationRatesFromGenomicData][Mutation Rates from Genome Resequencing]] %BR% How to estimate mutation rates (and confidence limits) from counts of mutations in multiple genome data sets. * [[FlowCytometry][Generic Flow Cytometry guidelines]]<br>Guidelines for flow cytometry on the Fortessa (basic) instrument. ---++ Experimental Evolution * [[ProceduresLongTermCompetitions][Competitive Fitness Assays]] <br>Measure the head-to-head relative fitness of two strains in co-culture. * [[ProceduresCompetenceAssays][Competence Assays]] <br>Measure the transformation frequency in _Acinetobacter baylyi_. * [[ProceduresEvolvabilityRiboseSubpopulations][Identifying Ribose Operon Deletions]] * [[ProcedureEvolvabilityMutationAccumulation][Mutation Accumulation for Microsatellite Strains]] * [[ProcedureBacterialMutationAccumulation][Bacteria Mutation Accumulation Experiments]] %BR% Propagate bacteria through single-cell bottlenecks to measure mutation rates. ---++ Nucleic Acid In Vitro Selection * [[ProceduresSpectrophotometricNucleicAcidQuantitation][Spectrophotometric Nucleic Acid Quantitation]] %BR% Determine concentrations of oligonucleotides and other nucleic acids from absorption spectra. * [[ProceduresPolyacrylamideGelElectrophoresis][Polyacrylamide Gel Electrophoresis]] %BR% Denaturing and nondenaturing (native) polyacrylamide gel electrophoresis. ---++ Working with non-canonical Amino Acids (ncAA) * [[ProtocolsncAAWorkingSolutions][ncAA Working Solutions and General Tips]] Working concentrations for common ncAA used here and a few tips for their use in Amberless strains. * [[ProtocolsncAAGFPIncorporationAssay][GFP Induction to test ncAA Incorporation]] Test incoporation of your ncAA synthetase using this GFP based protocol. ---++ Useful Resources * %ICON{airplane}% [[ProtocolsRetired][Retired and rarely used protocols]] * %ICON{help}% [[ProductManualLinks][Links to Product Manuals]]%BR% * [[http://openwetware.org/wiki/Electrocompetent_cells][OpenWetWare]] a Wiki of biological protocols * [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570007/][Enzymes used in molecular biology: a useful guide]]
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Topic revision: r138 - 2015-06-09 - DanielDeatherage