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:
  • 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
    • Koch Flow Core
      • Sony MA-900
        • Startup
        • Shutdown
    • Running Genomic pre-analysis using SnakePipes in Supercloud
    • Animal Facility
    • 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
      • Homemade EU click
        • Stock solutions
        • Add EU to cells
        • Dissociate
        • Fix cells
        • Permeabilize
        • Prepare click/label mix
        • Label and wash
      • HIVE scRNA-seq
        • Sample capture
        • HIVE processing
      • Immunofluorescent Staining
        • Solutions Required
        • Adherent Cell Staining
        • Cell Staining for Flow
      • 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
      • 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
      • sci-ATAC-seq
        • Background
        • Preparation (N days before)
        • Day 1: cell collection and fixation
        • Day 1: transposition
        • Day 2: library amplification
        • Day 3: library quantification
      • 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:
      • Titration of Tn5 transposition in MEFs
        • Part 1. Assembling the Transposase
        • Part 1.1 Prepping the Tn5 concentrations from Assembled Tn5 transposome per Transposition reaction (50uL)
        • Part 2. Transposition Reaction of Isolated nuclei
      • 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 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
      • 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
        • Confirm and purify
      • 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
      • Creating a new IVT template plasmid - NEED TO UPDATE
      • modRNA synthesis
      • IVT reaction
      • MluI digestion for pKG3198 (polyA encoded on plasmid)
      • 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)
        • 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
        • 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
        • Transduction with concentrated virus
        • Plat-E Retroviral Production
        • VSV production
        • AAV production in HEK293T
        • Viral Titer Calculation
  • Recipes
    • Bacterial recipes
      • LB selection plates
      • Antibiotic Stock Recipes
        • 1000X Ampicillin (Amp)
        • 1000X Kanamycin (Kan)
        • 1000X Chloramphenicol (Chlor)
      • Competent cells (NEB stable)
        • Day 1. Culture NEB stable cells for overnight.
        • Day 2. Grow the competent cells and stock the cells.
        • Day 4. Confirm the 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
      • 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
      • 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)
        • 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?
  • Bootcamp
    • IAP Bootcamp
      • 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
    • 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
    • “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
  • 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
  • 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
  • Protocols
  • in vitro transcription
  • View page source

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
  • Creating a new IVT template plasmid - NEED TO UPDATE
  • modRNA synthesis
  • IVT reaction
  • MluI digestion for pKG3198 (polyA encoded on plasmid)
  • IVT using MluI-digested pKG3198 (polyA encoded on plasmid)
  • mRNA transfection
  • MessengerMax transfection protocol
  • StemMACS transfection protocol

© Copyright 2023, Galloway Lab. Shared under the Creative Commons 4.0 Attribution International license. Last updated on Jan 23, 2023.

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