Protocols

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Contributing protocols Add new protocols Add a new lab- or person-specific protocol Add a consensus protocol (?) Improve protocols in development Please Contribute! Pulse-chase protein production DNase Protocol Etchevers:ChIP francais DNA Precipitation Addition of 3' A overhangs to PCR products Baby cell column Chromosomal DNA isolation from E. coli Protocols isolate cancer microparticles	In vivo Acetobacter xylinum [show][hide] Acetobacter Xylinum Culture Genomic miniprep/Sigma kit Arabidopsis thaliana [show][hide] Transformation Protocol for Arabidopsis Arabidopsis gDNA isolation Rapid plant gDNA extraction Escherichia coli [show][hide] Preparing chemically competent cells Electrocompetent cells Miniprep Electroporation Colony PCR Bacterial cell culture Beta-Galactosidase Assay (A better Miller) Miniprep/Qiagen kit Miniprep/Kit-free high-throughput protocol Transforming chemically competent cells Bacterial transformation Making a long term stock of bacteria P1vir phage transduction Preparing chemically competent cells (Inoue) Transforming chemically competent cells (Inoue) One step 'miniprep' method for the isolation of plasmid DNA TOP10 chemically competent cells Miniprep/GET buffer Maxiprep of plasmid DNA from E. coli Recombineering/Lambda red-mediated gene replacement ChIP-Chip E. coli Engineering BioBrick vectors from BioBrick parts/Colony PCR Cell counting/plating Cell and tissue lysis hub Cell cycle analysis In-fusion biobrick assembly Miniprep/TENS miniprep Knight:Colony PCR Endy:Colony PCR Endy:E. coli Western Blot Endy:STN Chemostat Protocol Knight:Preparing electrocompetent cells Knight:Electroporation Belcher/Knight: Electrocompetent Cells Lory:Quick transformant screen Seth:Quick Transformant Screen Sauer:Lysing E. coli with Lysozymes Knight:DNA Spots Knight:Miniprep low copy plasmids Knight:Beta-galactosidase assay Knight:Beta-galactosidase assay/96 well format Knight:Beta-galactosidase assay/Fluorescence Knight:Preparing stabs Duffy:LIC Cloning Huang: Agarose Slides for Imaging Richard Lab:Electroporation of E. coli Richard Lab:Preparing electrocompetent cells Erman's Lab:DNA Miniprep with Alkaline Lysis McClean:Competent Cells Gill:Rapid bacterial DNA prep Lactic Acid Bacteria [show][hide] Lactobacillus transformation (Serror 2002) Lactobacillus transformation (Kim 2005) Lactobacillus culture Lactobacillus miniprep Lactobacillus planarum miniprep Electro-transformation of Lactobacillus spp. In vitro modification of DNA for L. plantarum Lactobacillus transformation (Berthier 1996) Lactobacillus chromosomal integration Lactococcus transformation Lactobacillus transformation (Speer 2012) Mesoplasma florum [show][hide] Mesoplasma florum:Genomic DNA Mesoplasma florum:Electroporation Mesoplasma florum:Inverse PCR Transposon location Mesoplasma florum:Transposome construction Mesoplasma florum:restriction enzyme tests Mesoplasma florum: Tn5 Transposase Mesoplasma florum:RNA Purification Mouse [show][hide] Mouse keratinocyte cultures Isolation of murine splenocytes Intracellular cytokine staining for flow cytometry (mouse) Mouse tissue lysis for genotyping Tamoxifen administration to mice DNA extraction from tissue Cell and tissue lysis hub Sex genotyping of mice Mycobacterium smegmatis [show][hide] McKinney:TransformByElectroporation Plasmodium falciparum [show][hide] Saponin Lysis of RBCs Immunofluorescence Microscopy (Pf, live) Immunofluorescence Microscopy (Pf, fixed) Streptomyces [show][hide] Streptomyces:Protocols/Conjugation Streptomyces:Protocols/Mini-Maxi Prep Streptomyces:Protocols/PCR Streptomyces:Protocols/Spore Prep Streptomyces:Protocols/Transformation by Electroporation Cell and tissue lysis hub T7 [show][hide] Studier Lysate Prep Cesium Chloride Purification of T7 Gill:Making plate lysates Gill:Phage genomic DNA extraction Gill:Plating out phage Yeast [show][hide] High Efficiency Transformation Yeast DNA Prep Yeast Colony PCR Cell Cycle Arrest Layered plates Silver: Lysate for Western Silver: Colony PCR Silver: Time-Lapse Microscopy Assaying mating Dropout plates for yeast Endy:Yeast Colony PCR Preserving yeast cultures Freimoser:Protocols:FastYeastTransformation Spheroplast Transformation Cell and tissue lysis hub Smolke:Protocols/Western Smolke:Protocols/Library transformation McClean: Sporulation McClean: Colony PCR (Yeast) McClean: Alpha Factor Stock McClean: Cycloheximide Stock Solution McClean:Magic Marker Medias McClean:SC Media McClean: Genomic DNA Prep (Bust 'n' Grab Protocol) McClean: Glycerol stocks (yeast) McClean: Sequencing Colony PCR Product SDS-PAGE sample buffer (Morris formulation) McClean: Plasmid Loss Assay McClean: Fixation of Yeast (Bisaria Protocol) McClean: Fixation of Yeast (P. Xu Protocol) McClean:Making and Using Frozen Yeast Competant Cells McClean:SC Canavanine McClean: Fixation of Yeast (McClean Protocol) McClean:Genomic DNA prep (LiAc SDS method)	In vitro Nucleic acids [show][hide] Agarose gel electrophoresis Phenol/chloroform extraction Ethanol precipitation of nucleic acids Quantitating nucleic acids PCR techniques Real-time PCR QRT-PCR Q-PCR Bioanalyzer Alm:Agarose gel electrophoresis DIYbio:Notebook/Keiki Gels DNA [show][hide] Agarose gel electrophoresis DNA ligation Yeast DNA Prep Ethanol precipitation of small DNA fragments Purification of DNA Annealing primers PNK Treatment of DNA Ends Miniprep Designing primers Phosphatase treatment of linearized vector Site-directed mutagenesis Restriction digest Phenol/chloroform extraction Sequencing DNA PCR Ethanol precipitation of nucleic acids 'Round-the-horn site-directed mutagenesis Library Generation Resuspension of primers Miniprep/Qiagen kit Miniprep/Kit-free high-throughput protocol One step 'miniprep' method for the isolation of plasmid DNA Cloning DNA Synthesis from Oligos PCR Overlap Extension Miniprep/GET buffer Electrophoretic mobility shift assay Centrifugal filtration/Nucleic acids Affymetrix DNA labelling for gene expression arrays Annealing and primer extension TempliPhi Quantitating nucleic acids Maxiprep of plasmid DNA from E. coli PCR techniques Real-time PCR QRT-PCR Anneal Extend PCR Protocol - for linking 2 pieces of DNA Streptavidin purification of DNA fragments DNA Precipitation Q-PCR Cloning and sequencing Engineering BioBrick vectors from BioBrick parts/DNA ligation Engineering BioBrick vectors from BioBrick parts/Dephosphorylation Engineering BioBrick vectors from BioBrick parts/Restriction digest Assembly pcr Cloning Checklist Bioanalyzer Size selective DNA precipitation SB DNA blot (Southern) Polyacrylamide gel electrophoresis DNA Seq Prep Cloning Protocol DNA Quantification Rapid plant gDNA extraction Amplified insert assembly Miniprep/TENS miniprep MIT:Sequencing BioBrick DNA Endy:Templiphi Knight:Micropure EZ and Microcon purification Knight:Annealing and primer extension with Klenow polymerase Knight:DNA ligation using NEB Quick Ligation Kit Knight:Restriction Digest Knight:Site-directed mutagenesis/Single site Silver: PCR Knight:Agarose gel electrophoresis Knight:TOPO TA cloning Endy:Double stranding oligo libraries Knight:Sequencing DNA Knight:Annealing and primer extension with Taq polymerase Knight:Reconstituting primers Kafatos:Minipreps "Core Sample" PCR: Amplifying DNA from agarose gels Knight:Electrophoretic mobility shift assay Jamesh008:consensus DNA ligation protocol Lory:Quick transformant screen Knight:Maxiprep Knight:PhoenIX Maxiprep Kit Seth:Quick Transformant Screen Knight:TempliPhi Sauer:Stitching Genes by PCR Streptomyces:Protocols/DNA Digest Etchevers:ChIP francais Knight:Site-directed mutagenesis/Multi site Knight:Miniprep low copy plasmids Alm:Agarose gel electrophoresis Knight:DNA ligation using T4 DNA ligase NanoBio: PCR Sacks:RAD-seq Duffy:LIC Cloning Weiss Lab:DNA labeling DIYbio:Notebook/Keiki Gels Richard Lab:Agarose Gel Electrophoresis Restriction Digest: Partial Richard Lab:Restriction Digest Richard Lab:Amplified insert assembly Corum:PCR Corum:digest Corum:T4 Ligation Corum:DNA Digestion Corum:DNA Hybridization Corum:Digestion Corum:Whole Plasmid PCR Brennan:Miniprep for vaccinia virus DNA isolation RNA [show][hide] Agarose gel electrophoresis RNA extraction Probe Prep, 32P End-Labeled Probes In vitro transcription with T7 RNA polymerase Phenol/chloroform extraction Ethanol precipitation of nucleic acids RNase Protection Assay Northern Blot, 32P End-Labeled Probes RNA Half-life Affymetrix One Cycle Eukaryotic Gene Expression Sample Processing RNA Quality Control Affymetrix Target Hybridization Affymetrix Two Cycle Eukaryotic Gene Expression Sample Processing RNA electrophoresis Quantitating nucleic acids PCR techniques Real-time PCR QRT-PCR/Single tube QRT-PCR QRT-PCR/Two tubes Q-PCR RNA extraction using trizol/tri Bioanalyzer RNA blot (Northern) Making RNA probes with T7 transcription Endy:Northern Blot, 32P End-Labeled Probes Endy:RNA extraction Knight:In vitro transcription Knight:RNA electrophoresis/Denaturing Knight:RNA electrophoresis Knight:RNA electrophoresis/Native Etchevers:Extraction ARN francais Sauer:RNA Purification from E. coli Alm:Agarose gel electrophoresis Endy:Northern blot, AlkPhos end-labeled probes Dahlquist:DNA Microarray Protocol Griffin:Nested RT-PCR DIYbio:Notebook/Keiki Gels Protein [show][hide] Beta-galactosidase assay Acrylamide gels Silver staining Purification of His-tagged proteins Free Sulfhydryl Determination Antigen retrieval Electrophoretic mobility shift assay Protein blot (Western) hub Chromatin Immunoprecipitation Dot Blot User:Leprevost/Purification of His-tagged proteins/Denaturing Bioanalyzer Isolate leydig cells from testes Lactobacillus planarum miniprep Protein Purification - Salting Out Feruloyl Esterase Protocols Beta-galactosidase Screen Beta-glucuronidase protocols User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/23 SDS-PAGE sample buffer (Morris formulation) Endy:E. coli Western Blot Sauer:Expression/purification of 35S-Met proteins Silver: Lysate for Western Silver: Coomassie Stain Silver: Destain Keating:Experimental Protocols:HPLC Knight:Purification of His-tagged proteins/Denaturing Knight:NuPAGE electrophoresis Knight:Electrophoretic mobility shift assay Knight:Purification of His-tagged proteins Knight:Purification of His-tagged proteins/Native Knight:Protein solubility Knight:Purification of His-tagged proteins/Denaturing with refolding Knight:Centrifuge desalting Knight:Centrifuge desalting/Zeba columns Knight:NuPAGE electrophoresis/Fast staining Knight:NuPAGE electrophoresis/Slow staining Knight:NuPAGE electrophoresis/Hybrid staining Sauer:Lysing E. coli with Lysozymes Knight:NuPAGE electrophoresis/Gel drying Etchevers:ChIP francais Alm:Agarose gel electrophoresis ISISBio:Protocols/p11 resin preparation ISISBio:Protocols/Sortase mediated ligation ISISBio:Protocols/Sortase mediated ligation/Small molecule ligation ISISBio:Protocols/Sortase mediated ligation/Solid support ligation ISISBio:Protocols/Sortase mediated ligation/Protein DNA ligation Griffin: Ultimate Immunoprecipitation Guide Griffin:Nuclear Extract Preparation Griffin:Western Blot Optimization Griffin:Immunohistochemistry Paraffin Griffin:Membrane Stripping and Reprobing Griffin:Antibody Elution Buffers Griffin:In Vitro Kinase Assay Griffin:Antibody Related Solutions & Recipes Griffin:Isolate Leukocytes from whole blood Fong:Bradford protein assay Prbbbb:small scale expression v1 Prbbbb:Coomassi Ethanol stain Prbbbb:inclusion body solubilization v1 Prbbbb:in vitro FRET FRB FKBP v1 Lipid [show][hide] Polar Lipid Analysis Mammalian cell culture [show][hide] Marek: Freeze-down/Thaw Griffin:Dual Immunofluorescence Cell Staining Griffin:Puromycin Selection Griffin:shRNA Transfection TissueCulture:splitting cells TissueCulture:Thawing cells Plant specific protocols [show][hide] Hop DNA Isolation Rapid plant gDNA extraction Transformation Protocol for Arabidopsis Transformation Protocol for Tobacco
	In silico Cloning [show][hide] Wikiomics:Cloning in silico Wikiomics:Site Directed Mutagenesis Data analysis [show][hide] FBA MoMA Coot and WinCoot for X-ray Crystallography Model Building Bionumerics Wikiomics:Bioinfo tutorial Wikiomics:ChIP-chip Wikiomics:Crystallography toolbox Wikiomics:Exon microarrays Wikiomics:Protein mass spectrometry Wikiomics:Protein volume Wikiomics:Staden Wikiomics:Viewing and sharing genome annotations Wikiomics:Pathway analysis Wikiomics:Genome annotation Wikiomics:Repeat finding DataONE:Protocols/Find GEO reuses DataONE:Protocols/Scopus citation counts from PMIDs Databases [show][hide] Wikiomics:PDB Wikiomics:Phylogenetic databases Wikiomics:RNA databases Tregwiki:Collected regulatory elements Modelling [show][hide] Modeller Homology Modelling Coot and WinCoot for X-ray Crystallography Model Building SynBioSS Wikiomics:Docking toolbox Sequence analysis [show][hide] BLAST Protein motifs Plasmapper Wwwblast Wikiomics:BLAST Wikiomics:FASTA Wikiomics:Multiple sequence alignment Wikiomics:Percentage identity Wikiomics:Phylogenetic databases Wikiomics:Protein function prediction Wikiomics:PSI-BLAST Wikiomics:RNA gene-finding Wikiomics:RNA homology search Wikiomics:RNA phylogenetics Wikiomics:RNA secondary structure prediction Wikiomics:Sequence motifs Tregwiki:Collected regulatory elements Wikiomics:BLAST tutorial Wikiomics:DNA sequencing Wikiomics:RNA splicing Wikiomics:SAGE Wikiomics:Orthologues Wikiomics:Mutation/SNP detection Structure analysis [show][hide] Coot and WinCoot for X-ray Crystallography Model Building Wikiomics:Docking toolbox Wikiomics:PDB Wikiomics:Protein volume Wikiomics:RNA databases Wikiomics:RNA gene-finding Wikiomics:RNA phylogenetics Wikiomics:RNA secondary structure prediction Transcriptional Regulation	Other General resources [show][hide] Current Protocols Most comprehensive source of protocols ranging from molecular biology to neuroscience. Current Protocols in Molecular Biology JoVE Journal of Visualized Experiments: An online research journal for publishing visualized (video-based) biological experiments. Protocol-online Useful protocols and a popular discussion section. Springer Protocols Aseptic Technique [show][hide] Flow cytometry [show][hide] Endy:FACS calibration with beads Synthetic Biology:BioBricks/Standard FACS protocol Endy:FACS protocols Shreffler:CFSE Labeling Shreffler:Basophil Activation Shreffler:BioCSI Basophil Activation Shreffler:Milk Program Shreffler:Basophil tSyk Shreffler:JAX Basophil Activation Shreffler:JAX Tcell studies Shreffler:Cisplatin Allergen Shreffler:Basophil Sensitization Shreffler:Eosinophil Isolation Shreffler:SCIT Basophil Activation Shreffler:Cryopreservation Microfluidics [show][hide] SortoStat/Operation Design a Microfluidic Chip Order a Microfluidic Chip from the Foundry Micado Tutorial Mathies:Valves Mathies:Drilling a Glass Microfluidic Wafer Mathies:Mask Making Mathies:Wafers Cleaning and Deposition Mathies:Photolithography Mathies:RIE Mathies:Glass Etching Mathies:Metal Deposition Mathies:Metal Etching Mathies:Glass Bonding Mathies:Nitride Mathies:Polyacrylamide Derivitization Mathies:Dynamic Coating Mathies:Sieving Matrices Mathies:Separation Buffer Mathies:Simulations Mathies:Introduction to Cell Culture Mathies:Culture in Broth Mathies:Calibration Curves Mathies:E COLI PCR Mathies:Glass Etching in BNC Mathies:RIE in BNC Mathies:Photolithography in BNC Mathies:Drilling in BNC Mathies:Back Door DropBase DropBase:Contact DropBase:Devices DropBase:droplet generation 4 inlets DropBase:droplet generation 3 inlets DropBase:droplet generation 2 inlets DropBase:droplet generation 3 inlets short delay DropBase:droplet generation 3 inlets long delay DropBase:droplet electrosorting DropBase:droplet electrosorting 2 DropBase:AADS DropBase:DLDS DropBase:Droplet generation 2 inlets DropBase:Picoinjection DropBase:Droplet sorting DropBase:Droplet 3 inlets DropBase:FFD-70-75 DropBase:ECD2 DropBase:ECD DropBase:TCD Dropbase: Double FF device Dropbase: Triple FF device Dropbase: Devices for 20 µm double emulsions Dropbase: Double-emulsion-01 Dropbase: Droplet generator Dropbase: Picoinjector Dropbase: AADS sorter Dropbase: Double-emulsion-02 DropBase:droplet electrosorting 3 DropBase:droplet electrosorting 4 DropBase:droplet electrosorting 5 Dropbase: 3-pL-droplets-01 Dropbase: VASA generator Dropbase: VASA 1st picoinjecton Dropbase: VASA 2nd picoinjecton DropBase:Fluidic connector Dropbase: 35um bead generator Dropbase: agarose bead generator DropBase:agarose bead generator Dropbase: spinDrop sorter Dropbase: spinDrop picoinjector DropBase:Anchored droplet DropBase:Sorter and FF devices DropBase:Picoinjection chip Microscopy [show][hide] Agarose Pads for Microscopy Fixing cells Methods to determine the size of an object in microns Silver: Time-Lapse Microscopy Koch Lab:Protocols/Microsphere-DNA tethering File:Low tech flow cell and wicks.JPG File:Low tech flow cell.JPG File:Low tech flow cell changing buffer with wick.JPG Koch Lab:Protocols/Microsphere-DNA tethering/Glass, dig, biotin, microsphere, 4kb DNA/Cleaning glass Paraffin embedding and sectioning Immunofluorescence Microscopy (Pf, live) Immunofluorescence Microscopy (Pf, fixed) TE antigen retrieval and immunostaining Mounting medium Nissl staining Plate reader [show][hide] Plate reader usage protocols Miscellaneous [show][hide] Endy:Pouring plates Endy:Pouring LB plates from prepped media Knight:pH meter/Calibration Knight:pH meter/Measurement Alm:Protoplasting Cell counting Category:Chromatography Category:Synchronization Category:Mammalian cell culture Category:Agarose gel electrophoresis Category:Human Category:Buffers Category:Alm Affinity purification of antibodies Category:Biomass Analysis InteLAB Anthrone cellulose quantification McClean: Tetrad Dissection McClean: Protocol Template McClean: Frogging Tetrads McClean: Yeast Mating Halo Assay McClean: NIS Elements Repair McClean: Quickie ImageJ Quantification McClean: Cleaning Floating Pin Replicators Shreffler: SDS-PAGE McClean: Yeast Recombinational Cloning IGEM:Virginia 2012/protocols/plaqueassay IGEM:Virginia 2012/Protocols/Plaque Assay IGEM:Virginia 2012/Protocols/Phage Amplification IGEM:Virginia 2012/Protocols/Phage Isolation IGEM:Virginia 2012/Protocols/Phage Replication Rate Assay IGEM:Virginia 2012/Protocols/Preparing Plates and Liquid Media IGEM:Virginia 2012/Protocols/PEG/NaCl Preparation IGEM:Virginia 2012/Protocols/SM Buffer Preparation IGEM:Virginia 2012/Protocols/Bordet Gengou (BG) Agar Plate Preparation IGEM:Virginia 2012/Protocols/SSM Media Preparation IGEM:Virginia 2012/Protocols/Preparing a Pertussis Culture IGEM:Virginia 2012/Protocols/LB Broth or Agar Preparation IGEM:Virginia 2012/Protocols/Isolating the Phage Genome IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells IGEM:Virginia 2012/Protocols/Passaging Pertussis Using the NanoDrop IGEM:Virginia 2012/Protocols/Using the NanoDrop McClean: Lab Database McClean:Phluorin Calibration Half-Inch Cartridges Using the Tube Cutter McClean: GEV Strain Construction McClean: Dry Ice-Ethanol Bath McClean: Takara PrimeStar PCR McClean: Flow Cells McClean: Anneal and Extend McClean: Pouring Gels for Electrophoresis Gill:TNA (Tryptone Nutrient Agar) Gill:TNB (Tryptone Nutrient Broth) Gill:TNA-SA Gill:Tris-HCl Gill:Lambda diluent (SM buffer) Gill:10x Lambda diluent (no gelatin) Gill:TSB (Tryptone Soy Broth) Gill:TSA (Tryptone Soy Agar) Gill:LB broth Gill:LB agar Gill:S liquid medium Gill:T-top (0.5% agar) Gill:10 X M9 salts Gill:10 X M9 salts + glucose Gill:M9 agar Gill:Supplemented M9 agar (M9sup) Gill:Supplemented M9 broth (M9sup) Gill:Vogel-Bonner glucose (VBG) minimal medium Gill:50X Vogel-Bonner salts McClean: Frogging a Serial Dilution McClean:Western Blot LAB Media Gill:Making frozen permanents McClean:Membrane stripping Gill:Confirming phage via gel electrophoresis McClean: FCS2 for Cycling Gill:Chung cells McClean:Blue Light Overview McClean:Annealing Oligos McClean:Oligonucleotide phosphorylation, Annealing and Ligation Gill:S. aureus genomic DNA isolation McClean:Invasive growth assay Gill:Subculturing phage isolates Gill:Phage enrichment Gill:Preparing phage specimens for TEM Gill:Calcium chloride competent cells Gill:S. aureus pre-treatment for plasmid preps McClean: Working with the LoxP/Cre System in S. cerevisiae McClean:E. coli Electroporation McClean:Membrane Stripping Mild Gill:Spot complementation test McClean: Intro to yeast McClean: Hemacytometer protocol for yeast McClean: PlasmidPrep QiagenKit McClean: Nanodrop2000 McClean: RollerDrum BrushReplacement McClean:BlueLightSetup McClean: Golden Gate-Making a new part McClean: Designing "Yeast Toolkit" compatible primers McClean: Golden Gate- Making a multi-part plasmid McClean: Sonication Yeast McClean: PlasmaPreen ORingReplacement McClean: LowDiskSpace QX200 Digital Droplet PCR McClean: Laboratory Cabinet Maint Need help with protocols? 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