<!-- Preferences start here * Set PAGETITLE = Barrick Lab :: Protocols Preferences end here --> ---+ Protocols and Laboratory Reference * [[ProtocolUpdatesNeeded][Updates Needed]] ---++ Lab Stocks * [[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> Information about plasmids commonly used in lab. * [[ProtocolsStrainDatabase][Strain Database]] <br> How to search and enter samples in the lab database. * [[ProtocolsFreezingStrains][Freezing Strains]] <br> How to freeze and archive strain samples. * [[AmountofLabStocks][Lab Stocks]] <br> List of amount of common lab stock solutions/media to keep on hand. * [[ReferenceKeioASKACollection][Keio ASKA Collections]] <br> Information about finding and using these _E. coli_ strains. ---++ PCR and Sequencing * [[ProceduresPrimerDesign][Sequencing/Genotyping Primer Design]] <br> Design primers to validate mutations in evolved genomes. * 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://www.ncbi.nlm.nih.gov//genomes/geblast.cgi?gi=24777][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]] * 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]] * [[ProtocolsOrderingPrimers][Ordering Primers]] * [[ProceduresPrimerDatabase][Primer Database]] * [[ProtocolsResuspendingPrimers][Resuspending Primers]] * [[ProtocolsStandardPCR][Standard PCR]] * [[ProtocolsTaq][Which polymerase is right for me?]] * [[ProceduresOverlapPCR][Overlap PCR]] * [[ProceduresStandardAgaroseGel][Agarose Gel Electrophoresis]] * [[ProcedureDNASequencing][DNA Sequencing]] * [[ProtocolsUsingAPE][Using APE]] * [[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)]] ---++ Genetic Modification * [[ProceduresUVMutagenesis][UV Mutagenesis]] * [[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 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]] ---++ 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. * [[ProceduresPulsedFieldGelElectrophoresisMapping][Pulsed-Field Gel Electrophoresis (PFGE) Mapping]]<br>To validate large-scale chromosomal rearrangements, deletions, and duplications. * [[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. * [[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. * [[ProceduresCrushSoakPolyacrylamideGelElution][Crush-Soak Elution]]<br>Recover DNA or RNA samples from polyacrylamide gels. * [[ProtocolsEthanolPrecipitation][Ethanol Precipitation]]<br>Precipitate DNA or RNA samples to remove salts and concentrate. * [[ProceduresPrimerExtension][Primer Extension]] * [[ProceduresSelectionPCR][PCR for Nucleic Acid Selections]] ---++ Bioinformatics / Source Code Management * [[ProtocolsRNASeqDifferentialExpression][Analyzing RNA-Seq data]] <br>Analyzing RNA-Seq data for differential expression. * [[ProtocolsSettingUpAutotools][Setting up compilation using Autotools]] * [[ProtocolsUploadingDataToSRA][Sequence Read Archive Upload]]<br>Uploading sequence data and obtaining an accession number for sequencing data from the SRA database. * [[ProtocolsRunningBreseqOnTACC][Running <i>breseq</i> on TACC]] * [[ProtocolsBreseqResults][Interpreting <i>breseq</i> output.]] <br>How to interpret, check, and manually correct <i>breseq</i> results. ---++ Environmental Microbiology * [[ProtocolsSoilGenomicDNAPrep][Extracting Clean Metagenomic DNA From Soil]] ---++ Writing * [[ProtocolsStyleGuide][Barrick Lab Style Guide]] ---++ See Also * [[http://openwetware.org/wiki/Electrocompetent_cells][OpenWetWare]] a Wiki of biological protocols * [[ProtocolsRetired][Retired and rarely used protocols]] ---++ Misc. Lab Protocols * [[AutoclaveUse][Autoclave Use]] * [[GlasswareCleaning][Cleaning Glassware]] * [[RattlerBeads][Reusing plating beads]]
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Topic revision: r88 - 2012-04-06 - JeffreyBarrick