Galloway Lab Protocols
  • General Lab Maintenance
    • Replacing microcentrifuge motor mounts
      • Required materials
      • Procedure
    • Cleaning and autoclaving guidelines
      • Washing glassware and tools
        • Glass bottles and flasks
        • Glass beads
        • Dissection Tools
      • Autoclaving
        • Tip boxes
        • Tube containers
        • Glass pipette containers
        • Toothpicks
        • Flasks
        • Dissection tools
        • Glass bottles (containing liquid or glass beads)
    • Cleaning BSCs
    • Cleaning microcentrifuges
    • Regular computer maintenance
      • Installing Attune software updates
    • Checking data backups
      • Onedrive backups
        • Once a year checks
      • NGS backups
        • Adding a new NGS dataset
        • Once a year checklist
    • Incubator decontamination
      • Moana and Maui (Main TC)
      • Hei Hei and Pua (Main TC)
      • Te Kā (Quarantine) and Te Fiti (66-219)
    • Lab Safety Information
      • General Information
      • Room-Specific Information
    • OneDrive sync problems and replacing the logged-in user
      • OneDrive sync problem debugging
        • It still doesn’t work!
      • Unable to pin OneDrive folders to the quick access
      • User replacement
    • Ordering
      • Direct ordering through Coupa
      • Attaching a quote to a Coupa order
      • Requests through Quartzy
      • Bulk Plastics Order
    • Plasmid Database
      • Quartzy
      • Plasmid website
    • Receiving packages
      • Receiving orders in Coupa
    • Recycling
    • Remote desktop for lab computers
      • Enabling remote desktop
      • Remoting in to lab computers
        • Windows
        • MacOS
        • Linux
    • Deli fridge starter replacement
      • Background
      • Disassembly
      • Debugging
      • Assembly
      • Replacements:
    • Replacing shake incubator drive belts
      • Required McMaster parts
      • Procedure
  • Instrument Use and Core Facility Access
    • Attune operation
      • Attune startup and shutdown
        • Quick startup checklist
        • Quick shutdown checklist
        • Initial inspection and startup
        • Performance test
        • Shutdown
      • Attune operation
        • Procedure descriptions
        • Weekly system cleaning
        • Every ~3-6 month system decontamination
        • Focusing fluid refill
        • Full Attune guides
      • Attune software
        • Common processes
        • Creating an experiment
        • Customizing the workspace
        • Exporting data
      • User-replaceable parts
        • Main syringe
        • Sample Injection Probe (SIP)
        • Focusing fluid filters
      • Attune troubleshooting
        • Delay before events start running
        • Fluid leak from the autosampler SIP onto a running plate
        • Wells skipping and the autosampler fails to draw focusing fluid
        • Power cycling the Attune
    • Nikon operation
      • Microscope Components
      • Startup/Shutdown
        • Turning the power on
        • Turning the power off (last user of the day)
      • Simple single image acquisition
        • Sample setup
        • Basic microscope controls
        • Simple image acquisition and exporting
        • Cleaning up
    • Koch Flow Core
      • Sony MA-900
        • Startup
        • Shutdown
    • Running Genomic pre-analysis using SnakePipes in Supercloud
    • Animal Facility
      • Mouse Colony Manager Lab Job
      • How to submit a CAC amendment
      • Ordering a new mouse strain
      • Recovering cryopreserved embryos
    • Cluster Computing
      • Creating and setting up an account
        • First-time setup
        • Per-project setup
        • Transferring files
      • KL notes
    • Keck Microscopy Facility
    • Myco Testing
      • Getting Access
      • Submitting Testing Request in iLab
      • Submitting Samples for Testing
      • Results
    • Data Transfer of Sequenced files from BioMicro Center to Supercloud
    • MIT SuperCloud Access
    • Keyence beginner’s user guide
      • Basic Operation
      • Default image settings
      • 60X Magnification
        • Tips for cleaning
        • How to use the 60X lens
      • Multi-point capture
      • Z-stack
      • Stitching
    • Keyence Time Lapse Imaging
      • Setting up the incubation chamber
      • Using the Keyence Software
      • Tips and Tricks
  • Protocols
    • Biochemical and analytical protocols
      • Genomics and sequencing methods
        • Sequencing technologies
        • The basics
      • Homemade EU click
        • Stock solutions
        • Add EU to cells
        • Dissociate
        • Fix cells
        • Permeabilize
        • Prepare click/label mix
        • Label and wash
      • Bulk RNAseq with Plasmidsaurus
      • Immunofluorescent Staining
        • Solutions Required
        • Adherent Cell Staining
        • Cell Staining for Flow
      • Alkaline Phosphatase Staining
        • Solutions Required
        • Procedure
      • ATAC-see
        • Transposase buffer preparation
        • ATAC-see
      • CellBaum User’s Guide
        • Local Usage
        • MIT Supercloud Usage
      • CellTrace Dye Labeling
        • CellTrace dyes available in lab
        • Other materials needed:
      • Sample Prep and Confocal Imaging at the Koch Microscopy core
        • Confocal access and Experimental planning:
        • Confocal imaging
        • Saving and Export of files
      • Droplet Digital PCR (ddPCR)
        • Design
        • Protocol
        • References
      • HCR DNA-FISH
        • Fixation
        • Hybridization
        • Probe Preperation
        • Post-Hybridization
      • Halo-tag Labeling
        • Halo-tag cell permeable dyes available in lab
        • Other materials needed:
      • HCR RNA-FISH
        • Day 1
        • Day 2
        • Day 3
      • Live nuclear staining using Hoechst
        • Materials
        • Protocol
      • Image segmentation and particle analysis in ImageJ
        • Defining regions of interest (ROI)
        • Quantifying nuclear-localized foci
        • Merging images in ImageJ
      • KAPA SYBR® FAST qPCR
        • Protocol
        • 1. Master Mix Preparation
        • 2. Reaction Setup
        • 3. qPCR
        • 4. Data Analysis
      • Image analysis using the KI workstation - Nemo
        • LasX 3D Image Viewer
        • Imaris
      • Magnetic bead clean-up for genomics libraries
        • Uses
        • Determine Bead Volume to use by size range you wish to recover:
        • Preparations:
        • Basic Bead Clean Protocol:
        • 2-step “Big” bead clean to remove large fragments >~350bp.
      • Nuclei Isolation for Downstream Genomics Applications
        • Nuclear Isolation
        • Recipes
      • Psoralen-qPCR supercoiling assay
        • Experiment setup
      • Single cell qPCR
        • Single-cell acquisition
        • Lysis, reverse transcription (RT), and specific target amplification (STA)
        • Single-cell qPCR on Fluidigm Biomark
      • SingleShot RNA isolation and Reverse Transcription
        • Materials need for RNA isolation
        • Materials needed for cDNA synthesis
        • RNA isolation using SingleShot Cell Lysis
      • Snap-tag Labeling
        • Snap-tag cell permeable dyes available in lab
        • Other materials needed:
      • Western Blot
        • Lysis
        • Bradford Assay
        • Protein Gel Casting
        • Loading and Running the Gel
        • Coomassie Staining
        • Transferring the protein from the gel to the membrane
        • Antibody Staining
    • Cloning protocols
      • Cloning Workflow Overview
        • Step 1: Generate DNA fragments
        • Step 2: Assemble plasmids
        • Step 3: Transform bacteria
        • Step 4: Screen colonies
        • Step 5: Purify plasmid DNA
        • Step 6: Sequence
        • Step 7: Glycerol stock
        • Starting with an assembled plasmid
      • Cloning Workflow Timeline
        • Typical Timeline
        • Accelerated Timeline
      • Colony PCR
        • Materials
        • Protocol
      • For hard to amplify templates
      • Restriction digest
      • Gel electrophoresis and extraction of DNA
        • Gel electrophoresis
        • Gel extraction
      • Gibson assembly
        • Protocol
      • Glycerol Stocking
      • Golden Gate assembly
        • Protocol for PCR fragments
        • Protocol for pPV plasmids
        • Reference for pPV connector sequences
        • Reference for entire Golden Gate workflow
      • Ligation assembly
        • Protocol
      • LR cloning
        • Protocol
        • Expected results
      • Oligo annealing for ligation cloning
        • Oligo phosphorylation
        • Oligo annealing
        • Combined oligo phosphorylation and annealing
      • PCR
        • Q5
        • PrimeSTAR
        • Taq
        • KOD Xtreme
        • Confirm and purify
      • Plasmid Prepping
        • Miniprep
        • Midiprep
        • Maxiprep
      • Cloning tips, tricks, and troubleshooting
        • PCRs
        • Gibsons
        • Golden Gates
        • Generally difficult assemblies
      • Touchdown PCR
        • Touchdown PCR
      • Bacterial Transformation
        • Cloning Summary
        • Transformation (Chemically Competent Bacteria)
    • in vitro transcription
      • Design and preparation of DNA IVT template
        • Creating a new IVT template plasmid
        • Generating IVT linear DNA template from an IVT template plasmid
        • PCR-amplified template
        • MluI digested template
      • circRNA synthesis
        • Generating IVT linear DNA template from plasmid
        • IVT reaction for circRNA
      • modRNA synthesis
        • IVT reaction
        • IVT using MluI-digested pKG3198 (polyA encoded on plasmid)
      • mRNA transfection
        • MessengerMax transfection protocol
        • StemMACS transfection protocol
    • Protein production
      • Protein gel casting
        • Required stock solutions
        • Casting protocol
        • Gel casting setup
      • Chitin-binding protein purification
        • Required solutions
        • Protocol
        • Dialysis and concentration protocol
        • Regeneration Protocol
      • Denaturing protein gel run and staining
        • Solutions required
        • Running procedure
        • Staining (Coomassie)
      • Protein expression
        • Protocol
      • His-tag protein purification
        • Source
        • Required solutions
        • Protocol
    • TC protocols
      • Induced pluripotent stem cells (iPSCs)
        • STRAIGHT-IN Line Creation
        • Culturing Human iPSCs
        • Transient Transfection of iPSCs
      • Other TC Protocols
        • 293T Rogi2 single LP V4 (cKG087)
        • SNAP circuit
        • Cell Line Creation
        • Rogi1 and Rogi2 LPs
        • Electroporation of plasmids into MEFs
        • Murine Bone Marrow Isolation
        • Murine Splenocyte Isolation
        • Transgene expression control through ribozyme switch (96-well plate version)
      • Reprogramming
        • MEF-to-iPSC Reprogramming
        • Human Fibroblast to iMN Reprogramming
        • Laminin coating for human reprogramming
        • Specialized coatings for neurons
        • 1X Borate Buffer for TC
        • PEI coating for neurons
        • PLO coating for neurons
        • MEF-to-iMN Reprogramming
        • Hb9::GFP iMN sort
      • TC Basics
        • TC Best Practices
        • Adherent Cell Culture
        • Freezing and thawing cells
        • Flow Cytometry
        • Glass slide cell culture
        • Culturing Jurkat Cells
        • Transient HEK293T Transfection
      • Virus
        • Virus safety
        • Virus production in HEK293T cells
        • Transduction with concentrated virus
        • Plat-E Retroviral Production
        • VSV production
        • AAV production in HEK293T
        • Measuring viral titer
  • Recipes
    • Bacterial recipes
      • LB selection plates
      • Antibiotic Stock Recipes
        • 1000X Ampicillin (Amp)
        • 1000X Kanamycin (Kan)
        • 1000X Chloramphenicol (Chlor)
      • Competent cells (NEB stable)
        • Day 1. Start an overnight culture
        • Day 2. Prepare competent cells
        • Day 4. Check for cell growth
      • DNA wash buffer
        • DNA wash stock solution (5x)
        • DNA wash buffer
      • Bacterial media recipes
        • TB (Terrific Broth)
        • 10x TB salts
        • SOC Media
        • LB Media
      • Other Solutions
        • Glycerol (60%)
        • 1xTAE buffer
    • FISH recipes
      • FISH buffer recipes
        • Probe Hybridization Buffer
        • Probe Wash Buffer
        • Probe Amplification Buffer
        • 5X SSCT
    • TC recipes
      • DNase (2X) and papain (2X)
        • DNase and papain from 2X
        • L-Cysteine (50 mM)
        • Papain (2X: 1mM EDTA, 2mM L-cysteine, 40 U papain/mL)
        • DNase (2X: 4,000 U/mL solution)
      • FBS heat inactivation
      • Common aliquot sizes for TC
        • Neurotrophic factor stocks (10kx and 1kx)
        • RepSox (1kx)
        • Fetal Bovine Serum (FBS) Aliquots (50 mL)
        • Pen-strep aliquots (100X, 5 mL)
        • 50X Geltrex aliquots
      • TC media recipes
        • N3 media
        • MEF media
        • Breast cancer 1 media
        • Breast cancer 2 media
        • Breast cancer 3 media
        • Breast cancer 4 media
        • Epithelial cell 1 media
        • Epithelial cell 2 media
        • Glia media
        • Sorting/Collection media
        • Motor neuron dissociation media
        • Freezing media
        • HEPES-buffered DMEM
        • iPSC Reprogramming Media
      • Other TC Solutions
        • Phosphate buffered saline (PBS)
        • 0.1% Gelatin
        • EDTA (0.5M, pH=8.0)
      • PEI Stocks
        • Materials
        • Dissolve and Aliquot PEI
        • Test PEI Ratio
        • Reference
      • Small molecule stocks for TC
        • Antibiotic concentrations
        • Other stock and working concentrations
        • Indole-3-acetic acid (Auxin, IAA)
        • Indole-3-acetamide (Auxin precursor, IAM)
        • 50mM O6-Benzylguanine (O6BG)
        • 25mM Guanine
        • 50 μM AP1903/Rimiducid (1000x stock - old DSP)
        • 1 mM or 100 µM AP1903/Rimiducid (100kx or 10kx stock - current NBW)
        • Puromycin, 1 mg / mL (1,000x stock)
  • Plot Gallery
    • High-quality PDF figure export
      • Intro
      • Problematic features
        • Transparency/opacity
        • Gradients/blurs
      • Final figure export
        • Illustrator
        • Matplotlib/Python
      • Preflight
    • Flow plots with adjunct histograms
    • Histograms
      • Function to make subplots for several variables
    • Mixed distribution + mean plots
      • Gating plot
      • Box plot with well means
      • Violin plot with well means
    • Multiple-axes plots
    • Testing matplotlib directives
      • Subheading
    • Notes on reproducing examples
  • Writing Guide
    • Additional resources
      • Big picture resources
      • Getting into the finer details
    • Points of style
      • Specific and nonspecific subjects
      • Hyphenation
      • Numbers in text
      • How to write gene and protein names?
      • References
    • Guiding principles
      • Tips
      • Outline
      • Active Voice
      • Editing and revising
    • What is a paper?
  • Training
    • “How to” Training Series
      • Tutorial 1: How to Write a Proposal
      • Tutorial 2: How to Read a Paper
      • Tutorial 3: How to Review a Paper
      • Tutorial 4: Graphic Figure Design
      • Tutorial 5: How to Compose Figures
    • Gene expression modeling with ODEs
      • Tutorial 1: Modeling gene expression with differential equations
      • Tutorial 2: Examining parameters affecting our models
      • Tutorial 3: Michaelis-Menten Kinetics
      • Tutorial 4: Modeling repressors
      • Tutorial 5: Hill functions
    • Onboarding
      • Day 0: Software and training setup
        • EHS setup and trainings
        • Software and webservices
      • Day 1: First day in lab
        • Onboarding form
        • Lab-specific safety training
        • Lab citizenship
        • Core facility access
      • Day 2+: Content-specific trainings
        • Core lab techniques
        • Computational skills
        • Research reading and writing
      • Computational environment check
        • Initial environment check
        • Protocols check
        • Completion date
      • An introduction to Git
        • Acknowledgements
        • Motivation
        • Git by example
        • Basic Git terms
        • Downloading or starting a repository
        • Basic history terms and git status: What’s happening?
        • Branching out, adding, and checking out files
        • Snapshotting your work
        • Ignoring files
        • Recovering history: reverting commits and file-level checkouts
        • Working with others: remotes and inevitable merge conflicts
        • Conclusion
      • Software Tips and Tricks
        • One date format to rule them all/naming
        • Paper RSS feeds
        • Creating feeds from searches
        • Zotero
        • Better Quartzy
        • Regex help
        • YAML files
        • Fonts
        • Activate SnapGene remotely
      • Startup checklist when working with repositories
        • New repository (Python)
        • New repository (Julia)
        • New repository (R)
        • Existing, non-data-driven repository (e.g. protocols)
        • Existing, data-driven repository (e.g. tangles_model)
      • On Terminals and Shells
        • Motivation
        • The basics
        • Interface basics
        • Starting off at home
        • Moving away from home
        • Relative and absolute paths
        • File operations
        • Editing and viewing files
        • Finding things in documents
        • Exercise
        • Extras
  • Tech documentation
    • Builder config and update
      • Current CI jobs
      • Updating the CI job
    • Possible build/install errors
      • Virtual environment install errors
      • No PDF is generated when I push
      • My screenshots aren’t visible
    • Slack bot (labbot)
      • Accessing the labbot server
      • Updating labbot
        • Normal updates
        • Unusual updates
      • Label printing
    • Self-hosted data storage
      • NAS setup
      • Nextcloud setup
      • Network setup
      • Touchstone setup
      • Backup setup
  • Contributor guide
    • Local environment setup
      • Text editor
      • git
      • Python/Sphinx setup
    • Standard workflow
    • Local building
      • Local previewing
      • In case of build errors
    • Online editing through Github
    • Repository layout
    • Basics of reStructuredText
      • Simple markup
      • Explicit markup
      • Admonitions
      • Code
      • Tables
      • References and links
      • Images
      • Math
Galloway Lab Protocols
  • Tech documentation
  • View page source

Tech documentation

  • Builder config and update
    • Current CI jobs
    • Updating the CI job
  • Possible build/install errors
    • Virtual environment install errors
    • No PDF is generated when I push
    • My screenshots aren’t visible
  • Slack bot (labbot)
    • Accessing the labbot server
    • Updating labbot
    • Label printing
  • Self-hosted data storage
    • NAS setup
    • Nextcloud setup
    • Network setup
    • Touchstone setup
    • Backup setup

See the Builder config and update if you are interested in how the continuous build system works, or you’d like to fork this repository to make it your own.


© Copyright 2025, Galloway Lab. Shared under the Creative Commons 4.0 Attribution International license. Last updated on Sep 25, 2024.

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