<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 * [[LabNotebooks][Lab Notebooks]] <br />Best practices for planning and recording your science. * [[StandardMicrobiologicalPractices][Standard Microbiological Practices]] <br />aka the approximately Ten Commandments of microbiology lab research. * [[AutoclaveSterilization][Autoclave Sterilization]] * [[GlasswareCleaning][Cleaning Glassware]] <br />Including Small Flasks, Large Flasks, Test Tubes, and Plating Beads. * [[ProtocolsMediaRecipes][Media Recipes]] <br />Solid and liquid media for bacterial growth. * [[ProtocolsAntibioticStockSolutions][Antibiotics, Supplements, Non-canonical amino acids]] <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. * [[ProtocolsReagentsPfuSso7d][Pfu-Sso7d polymerase purification]] <br />How to express and purify 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. * [[Strain_side_notes][E. coli strains]] <br />Characteristics of _E. coli_ strains commonly used in the lab. * [[Microplate Reader Quick Start Guide][Microplate Reader Quick Start Guide]] <br />Quick guide with main steps for setting up and using the microplate reader in our lab. ---++ 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]] * [[https://www.benchling.com/primers/][Cloning / General Primer Design (Benchling)]] %ICON{external}% * [[ProtocolsOrderingPrimers][Ordering Primers]] | [[http://www.idtdna.com/ICMBSupplyCenter/Login.aspx][ICMB's IDT page]] %ICON{external}% | [[ProtocolsDegenerateBases][Degenerate bases]] * [[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]] <br> Simple and reliable method for introducing specific mutations into plasmids. ---++ Genetic Modification and Cloning * [[ProtocolsQuickChange][Site Directed Mutagenesis]] %BR% QuickChange-based protocol for site directed mutagenesis in plasmids. * Transformation: * [[ProtocolsElectrocompetentCells][Electrocompetent Cells]] * [[ProtocolsChemCompCellsHub][Chemically Competent Cells]] * [[ProtocolsConjugation][Conjugation]] * [[ProtocolsTestingTransformEff][Assessing Transformation Efficiency]] * [[ProtocolsUVLibrary][UV Mutagenesis]] %BR% Make a library of bacterial mutants with UV irradiation. * [[ProtocolsAraMarker][Selecting Arabinose Marker Revertants]] %BR% Direct selection of Ara+ revertants from Ara– strains. * [[ProtocolsColonyTransformation][Colony Transformation]] %BR% Rapid chemical transformation of E. coli colonies with high copy plasmids.. * [[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. * [[Golden Gate Assembly Protocols Main Page][Golden Gate Assembly Protocols]]<br/>Design and build constructs that are compatible with Golden Gate Assembly * [[ProtocolsP1Transduction][P1 Transduction]] <br />Quickly move mutations that are linked to a selectable marker between _E. coli_ strains ---++ 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 from OD600/platereader data and calculate growth rates with the Growthcurver R package. * [[ProtocolGrofit][Growth Rates with Grofit]]<br />Calculate growth rates with the Grofit R package. * [[ProtocolsPlasmidCopyNumber][Plasmid Copy Number]]<br />Measure copy number of plasmids compared to genome. * [[ProtocolsFluorescentProteinEvolutionaryStability][Fluorescent Protein Evolutionary Stability]]<br />Measure the evolutionary stability of fluorescent protein expression. ---++ Phage * [[ProtocolPhageLysate][Making Phage Lysates]]%BR%Procedure for _E. coli_ phages T4, T5, T6, and T7. * [[ProtocolsPhageTiters][Measuring Phage Titers]]%BR%Determine how many plaque-forming units are in a lysate. * [[ProtocolsPhageGenomicDNA][Isolating Phage Genomic DNA]]%BR%Purify DNA from a phage sample for sequencing or use as a PCR template. * T7 Phage Fitness Assays: * [[ProtocolsPhageAdsorptionRate][Measuring Adsorption Rate of T7 Phage]] * [[ProtocolsPhageLysisTiming][Measuring Lysis Timing of T7 Phage]] * [[ProtocolsPhageBurstSize][Measuring Burst Size of T7 Phage]] ---++ Genome Resequencing and Mutation Validation * [[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. * [[ProceduresKappaNgsPrep][Next-generation Sequencing Illumina library preparation]] %BR% Steps for generating Illumina libraries. * [[ProceduresPlasmidSequencing][NGS Plasmid Sequencing]] %BR% sample submission requirements and process overview for sequencing plasmids on an iSeq. ---++ Experimental Evolution * [[ProceduresLongTermCompetitions][Competitive Fitness Assays]] <br />Measure the head-to-head relative fitness of two strains in co-culture. * [[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. * [[ProcedureSelectingByEfficiency][Selection in Emulsions]] %BR% Using emulsions to select by yield ---++ 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]] ---++ Nucleic Acid Preparation, RNA-Seq, qPCR * [[ProtocolsEthanolPrecipitation][Ethanol Precipitation]] %BR% Precipitate DNA to remove salts * [[PhenolChloroformExtraction][Phenol/chloroform bacterial genome extraction]] %BR% Protocol for chemical extraction of very pure bacterial DNA * [[RNAPrep][Bacterial RNA isolation]]<br />Preparation of high quality RNA using RNA Snap method combined with Zymo column purification. * [[RNAPlantPrep][Plant RNA isolation]] * [[RNASeq][RNAseq]]<br />Preparation of rRNA-free libraries from bacterial culture for submission to GSAF core. * [[qPCR_hub][qPCR Hub]] * [[qPCR][Comparative RT-qPCR for differential gene expression]] * [[qPCR to quantify plasmid copy number][Absolute qPCR for plasmid copy number]] * [[Denaturing formaldehyde gels][Denaturing formaldehyde gels]]<br />Quick gels to verify size of RNA from e.g. transcriptions. Not suitable for purification. * [[ProceduresPolyacrylamideGelElectrophoresis][Polyacrylamide Gel Electrophoresis]] %BR% Denaturing and nondenaturing (native) polyacrylamide gel electrophoresis. * [[RNAT7][ _In vitro_ Transcription]] ---++ 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]] ---++ _in vitro_ Enzyme Activity Assays * [[ProtocolsBroccoliAptamerInVitroAssays][Measuring transcription _in vitro_ using fluorescent RNA aptamers (Broccoli, Spinach, etc.)]] * [[ProtocolsIntrocellularROSDetection][Intracellular ROS detection]] ---++ Working with Unusual Microbes * Bees: [[ProtocolsCulturingSnodgrassellaAlvi][<em>Snodgrassella alvi</em>]] | [[ProtocolsCulturingGilliamellaApicola][Gilliamella apicola]] | [[ProtocolsCulturingBartonellaApis][<em>Bartonella apis</em>]] | [[ProtocolsCulturingFirm_5][<em>Lactobacillus</em> "Firm-5"]] * Aphids: [[ProtocolsWorkingWithSerratiaSymbiotica][<em>Serratia symbiotica</em>]] | [[ProtocolsCulturingArsenophonusNasoniae][<em>Arsenophonus nasoniae</em>]] * Plants: [[ProtocolsWorkingWithPseudomonasSyringae][<em>Pseudomonas syringae</em>]] * Other: [[ProtocolsWorkingWithVibrioNatriegens][<em>Vibrio natriegens</em> (Vmax)]] | [[ProtocolsWorkingWithSerratiaMarcescens][<em>Serratia marcescens</em>]] * Yeast [[ProtocolsLithiumAcetateTransformation][<i>Saccharomyces cerevisiae</i>]] ---++ Working with Insects * [[ProtocolsAphids][Aphid Care and Protocols]] * [[ProtocolsLeafhoppers][Leafhopper Care and Protocols]] * [[ProtocolsHornwormCare][Tobacco hornworm (<em>Manduca sexta</em>) care]] ---++ _Acinetobacter baylyi_ ADP1 * [[ProtocolsAcinetobacterBaylyiADP1][ _Acinetobacter baylyi_ ADP1 Overview]] * 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]] * [[ProceduresCompetenceAssays][Competence Assays]] <br />How to confirm competence and measure the transformation frequency in _Acinetobacter baylyi_. * [[ProtocolsAcinetobacterTransformation][Transforming <em>Acinetobacter baylyi</em> ADP1]]<br />How to transform _Acinetobacter baylyi_ ADP1 with plasmids or PCR products. * [[ProtocolsAcinetobacterPuddleTransformation][<em>Acinetobacter baylyi</em> 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. * [[ProtocolsAcinetobacterGenomeManipulation][<em>Acinetobacter baylyi</em> ADP1 genome manipulation]]<br />How to make deletions, insertions, and other modifications to the _Acinetobacter baylyi_ genome. * [[ProtocolsAcinetobacterGoldenTransformation][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. * [[ProtocolsAcinetobacterOverlapPCRTransformation][ADP1 Overlap Extension PCR genome manipulation]]<br />How to make modifications to the _Acinetobacter baylyi_ genome with overlap PCRs. * [[ProtocolsAntibioticRemovalVerification][Quick 3hr Antibiotic Rescue Verification]]<br/>Same-day verification of antibiotic removal/"rescue" from counter-selection plates (e.g., AZT) * [[ADP1 Tn-seq Library Prep][ADP1 Tn-seq Library Prep]]<br />Protocol for preparing a Tn-seq library for ADP1 strains mutagenized with pBT20 ---++ 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: r257 - 2022-03-11 - JeffreyBarrick