<noautolink> <!-- Preferences start here * Set PAGETITLE = Barrick Lab :: Laboratory Protocols Preferences end here --> ---+!! Barrick Lab :: Laboratory Protocols %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]] * [[GlasswareCleaning][Cleaning Glassware]] * [[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. * [[ReferenceKeioASKACollection][Keio and ASKA Collections]] <br /> Information about finding and using these _E. coli_ strains. * [[ProtocolsFindStrainsPlasmids][Find strains and plasmids]] <br /> List of commonly used culture/plasmid collections. * [[ProtocolsFindChemicals][Find sources for chemicals]] <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]] | [[AcinteobacterBaylyiADP1QuickReference][Quick Reference]] * 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]] * [[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 * [[ADP1 Tn-seq Library Prep][ADP1 Tn-seq Library Prep]]<br /> Protocol for preparing a Tn-seq library for ADP1 strains mutagenized with pBT20 * [[qPCR to quantify plasmid copy number]] ---++ Genetic Modification and Cloning * [[ProtocolsConjugation][Conjugation]] General conjugation protocol for introducing plasmids to bacteria. * [[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 _E. coli_ stocks for transformation. * [[ProtocolsElectroporation][Electroporation of your Electrocompetent Cells]]<br /> Transform your Electrocompetent cells with DNA. * [[ProtocolsColonyTransformation][Colony Transformation]]<br />Rapid chemical transformation of E. coli colonies with high copy plasmids.. * [[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. * [[ProtocolsAcinetobacterGoldenTransformation][Acinetobacter baylyi ADP1 "Golden Transformation" genome manipulation]]<br />How to make fast near scarless deletions to the Acinetobacter baylyi genome using a modified Golden Gate Cloning methodology. * [[ProtocolsAcinetobacterPuddleTransformation][Acinetobacter baylyi ADP1 "in plate" and "puddle transformations"]]<br /> Transformation of ADP1 in solid medium is known to achieve higher transformation efficiency. Quick and ideal for difficult transformations or when low amounts of transforming DNA are available. * [[ProtocolsMegaprimerMegawhop][Megaprimer Megawhop Mutagenesis]] <br /> Simple and reliable method for introducing specific mutations into plasmids. * [[Protocols Golden Gate Assembly][Golden Gate Assembly (YTK version)]] <br /> Multi-part DNA assembly. * [[Protocols Golden Gate Making A New Part][Making a new Golden Gate Part Vector]] <br /> Cloning into the entry vector. * [[Protocols Golden Gate Assembly of Cassette Plasmids]][Assembly of Cassette Plasmids]] * [[ProtocolsCIDARMoclo][CIDAR Moclo Kit]] <br /> Using the CIDAR MoClo kit for golden gate assembly of plasmid variants * [[ProtocolsP1Transduction][P1 Transduction]] <br /> Quickly move mutations that are linked to a selectable marker between _E. coli_ strains. * [[ProtocolsChemCompCellsInoue][Preparing and Transforming Chemically Competent Cells]] <br /> No-kit Inoue method ---++ Mutation Rates, Growth Rates and Evolutionary Stability * [[ProtocolsFluctuationTests][Fluctuation Tests]]<br />Measure bacterial mutation rates to antibiotic resistance, phage resistance, or reversion of auxotrophy. * [[ProtocolsGrowthRates][Growth Rates]]<br />Measure bacterial growth rates from OD600/platereader data * [[ProceduresPhageFarming][Phage Stocks]]<br />Phage Lysate Preparation (T4,T5,T6) and Determining Phage Titer * [[ProtocolsFluorescentProteinEvolutionaryStability][Fluorescent Protein Evolutionary Stability]]<br />Measure the evolutionary stability of fluorescent protein expression. ---++ 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. ---++ 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. ---++ Flow Cytometry and Cell Sorting * [[FlowCytometry][Generic Flow Cytometry guidelines]]<br />Guidelines for flow cytometry on the Fortessa (basic) instrument. * [[ProceduresBacteriumCellSortingFACSAria][Sorting Bacterial cells using the FACSAria]] ---++ RNA Preparation, RNA-Seq, qPCR * [[RNAPrep][RNA Preparation]]<br />Preparation of high quality RNA using RNA Snap method combined with Zymo column purification. * [[RNASeq][RNAseq]]<br />Preparation of rRNA-free libraries from bacterial culture for submission to GSAF core. * [[qPCR][qPCR]]<br />Quantitative real time PCR for differential gene expression. ---++ 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. ---++ Non-canonical Amino Acids * [[ProtocolsncAAWorkingSolutions][Working with an Expanded Genetic Code]] <br />Properties of amino acids—both canonical and non-canonical—and their use in organisms with an expanded genetic code. * [[ProtocolsncAAStockSolution][Preparing Stock Solutions for ncAA]] <br /> Making stock solutions for non-canonical amino acids (ncAA). ---++ Protein Expression and Purification * [[ProtocolsLargeScaleProteinExpressionEColi][Large-scale Protein Expression in E. Coli]] * [[ProtocolsRunningSDSPAGEProteinGels][Running SDS-PAGE Protein Gels]] * [[Protocols6xHisTagProteinPurification][Purifcation of 6xHis-Tagged Proteins from E. Coli lysates]] ---++ Working with Unusual Microbes * [[ProtocolsCulturingSnodgrassellaAlvi][Culturing Snodgrassella alvi]] * [[ProtocolsCulturingGilliamellaApicola][Culturing Gilliamella apicola]] * [[ProtocolsCulturingBartonellaApis][Culturing Bartonella apis]] ---++ Accessing the Literature and Managing Citations and presentation information * %ICON{pdf}% [[ProtocolsOffCampusJournalAccess][Off Campus Journal Access]] <br />Methods of finding, discovering and accessing various journal articles * [[ProtocolsSavedArticleSearches][Saved Article Searches]] <br /> Set up automatic updates to find current publications of interest * [[ProtocolsMakingPresentations][How to make a good presenation]] <br />How to make a good presenation under different considerations. ---++ 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: r177 - 2017-02-06 - SimonDAlton