(r219) Barrick Lab :: Laboratory Protocols

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---+!! Barrick Lab :: Laboratory Protocols
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---++ 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.
   * [[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]]
   * [[ProtocolsOrderingPrimers][Ordering Primers]] | [[http://www.idtdna.com/ICMBSupplyCenter/Login.aspx][%ICON{external}% ICMB's IDT page]] | [[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]]
   * [[IlluminaSequencing][Illumina Next Generation Sequencing]]<br />Protocol for NGS library generation
   * [[<span style="color: #3a3a3a; font-family: Arial, Helvetica, sans-serif; font-size: 13px">T7</span><span style="color: #3a3a3a; font-family: Arial, Helvetica, sans-serif; font-size: 13px">mRNAsynthesis</span>][<span style="font-size: 13px; color: #3a3a3a; font-family: Arial, Helvetica, sans-serif">T7 </span><span style="font-size: 13px; color: #3a3a3a; font-family: Arial, Helvetica, sans-serif">mRNA synthesis</span>]]

---++ Genetic Modification and Cloning

   * [[ProtocolsConjugation][Conjugation]] %BR% General conjugation protocol for introducing plasmids to bacteria.
   * [[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&ndash; 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..
   * [[ProtocolsRestrictionEnzymeCloning][Restriction Enzyme Cloning]] %BR% Insert a target DNA sequence into a plasmid via restriction digest and ligation.
   * [[ProtocolsGeneGorgingMarker][Gene Gorging Neutral Markers]] %BR% &lambda; red genetic modification for mutations with a selectable or screenable marker using donor plasmid. For example, converting Ara+ strains to Ara&ndash;.
   * [[ProceduresGeneGorgingEvolvedAlleles][Gene Gorging Evolved Alleles]] %BR% &lambda; 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% &lambda; 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 />&lambda; 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.
   * [[ProtocolsMegaprimerMegawhop][Megaprimer Megawhop Mutagenesis]] <br />Simple and reliable method for introducing specific mutations into plasmids.
   * [[Golden Gate Assembly Protocols Main Page][Golden Gate Assembly Protocols]]
   * [[BroadHostRangeToolkit][Bee Microbiome Toolkit (BTK)]] <br />Golden Gate part kit derived from YTK for work in non-model bacterial species, particularly bacteria from the honey bee gut. 
      * [[Protocols Golden Gate Assembly of Cassette Plasmids][Assembly of Cassette Plasmid]]
   * [[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 
      * [[ProtocolsTransformingChemCompCells][Transforming Chemically Competent Cells]]
      * [[ProtocolsEfficiencyChemCompCells][Checking Transformation Efficiency of Chemically Competent Cells]]

---++ 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
   * [[ProtocolsFluorescentProteinEvolutionaryStability][Fluorescent Protein Evolutionary Stability]]<br />Measure the evolutionary stability of fluorescent protein expression.
   * [[ProtocolPhageLysate][Making Phage Lysates]]<br />E. coli phages T7, T4, others?
   * [[ProtocolsPhageTiters][Measuring Phage Titers]]
   * 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

   * [[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.
   * [[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

   * [[ProceduresSpectrophotometricNucleicAcidQuantitation][Spectrophotometric Nucleic Acid Quantitation]] %BR% Determine concentrations of oligonucleotides and other nucleic acids from absorption spectra.
   * [[ProtocolsEthanolPrecipitation][Ethanol Precipitation]] %BR% Precipitate DNA to remove salts
   * [[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][RT-qPCR for differential gene expression]]
   * [[qPCR to quantify plasmid copy number][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.

---++ Non-canonical Amino Acids

   * [[ProtocolsncAAWorkingSolutions][Working with an Expanded Genetic Code]] <br />Properties of amino acids&mdash;both canonical and non-canonical&mdash;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]]

---++ _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>]]
   * Other: [[ProtocolsWorkingWithVirbrioNatriegens][<em>Vibrio natriegens</em>]] | [[ProtocolsWorkingWithSerratiaMarcescens][<em>Serratia marcescens</em>]]

---++ _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]]
   * [[ProceduresCompetenceAssays][Competence Assays]] <br />Measure the transformation frequency in _Acinetobacter baylyi_.
   * [[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.
   * [[ProtocolsAntibioticRemovalVerification][Quick 3hr Antibiotic Rescue Verification]] 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

---++ Yeasty Protocols
   * [[ProtocolsLithiumAcetateTransformation][Lithium Acetate Transformation]]

---++ 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 presentation]] <br />How to make a good presenation under different considerations.
   * [[ProtocolsAccessNonelectronicJournalArticles][How to (legally) get a copy of inaccessible journal articles]] <br /> How to access journal articles where either (i.) UT doesn't have a subscription or (ii.) Article only exists as a physical copy

---++ 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]]
Barrick Lab > ProtocolList
Contributors to this topic
JeffreyBarrick, SeanLeonard, DanielDeatherage, PengGeng, CraigBarnhart, IsaacGifford, KateElston, GabrielSuarez, ColinBrown, SimonDAlton, AurkoDasgupta, BrianRenda, MichaelHammerling, MattMcGuffie, LindseyWolf, DaciaLeon, JordanMonk, ElizabethRobinson, AlvaroRodriguez, JuliePerreau, MarkKauth, JaredEller, WaldoDittmar, NeilGottel, TylerDeJong, SophiaLin, AnthonyVandieren, PraneshRao, LucioNavarro, MeghnaVergis, SteveSowa, XueZhang, CamiloGomez, EricBryan, DennisMishler, SarahBialik, AlexaMorton, StrattonGeorgoulis
Topic revision: r219 - 2018-06-07 - 19:31:50 - Main.ElizabethRobinson