Galloway Lab Protocols
latest
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
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
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
Colony PCR
Materials
Protocol
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
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
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
modRNA synthesis
IVT reaction
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
TC Basics
TC Best Practices
Adherent Cell Culture
Freezing and thawing cells
Transient HEK293T Transfection
Flow Cytometry
MEF-to-iMN Reprogramming
Human Fibroblast to iMN Reprogramming
Glass slide cell culture
Culturing Human iPSCs
Virus
Virus safety
Virus production in HEK293T
Transduction with concentrated virus
Plat-E Retroviral Production
VSV production
AAV production in HEK293T
Viral Titer Calculation
Rogi1 and Rogi2 LPs
Line summary
Integration into landing pad lines
SNAP circuit
Materials
Protocol
Cell Line Creation
PiggyBac (Sneha)
Day -1
Day 0
Day 1
Day 2-4
CRISPR (Deon)
Day -1
Day 0
Day 1
Day 2
Day 3
Days 4-8
TALENS (Chris)
Day -1
Day 0
Day 1
Day 2-4
Clonal selection or enrichment via limiting dilution
Day 5 - Week 2
Week 2 + 1 day
Week 3
Clonal selection or enrichment via flow sorting
Repicking
Genotyping your line
Rogi1 and Rogi2 LPs
Day -1
Electroporation of plasmids into MEFs
Overexpression
Plasmid Concentration Experiment
Transient Transfection of iPSCs
Calculations
Protocol
Transgene expression control through ribozyme switch (96-well plate version)
Materials
Day 1: Seed cells
Day 2: Transfection
Day 3 or 4
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
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
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
»
Bootcamp
»
“How to” Training Series
»
Tutorial 2: How to Read a Paper
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Tutorial 2: How to Read a Paper
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