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Megason Laboratory

Location: Harvard Medical School, Department of Systems Biology, 200 Longwood Ave., WAB536, Boston, MA 02115

Summary:

The Megason lab is interested in how the program contained in the genome is executed during development to turn an egg into an embryo. To this end we have initiated the Digital Fish project. In the Digital Fish project, we are using several technologies we developed including in toto imaging, GoFigure, and FlipTraps to "watch" the execution of circuits in living zebrafish embryos during development in a systematic fashion.

Affiliations:

People:

Resources:

Instruments

  • Eppendorf AG realplex2 real-time PCR machine ( Real-time PCR machine )

  • Leica MZ12.5 Stereomicroscope #1 ( Stereo microscope )

    With a Plan 1.0X objective, transmitted light bases, and a micromanipulator controlled Nanoject for microinjections. Lab contains six of these instruments.

  • NanoDrop 8000 Spectrophotometer ( Spectrophotometer )

    8-channel version of the Nanodrop. Capable of measuring DNA/RNA concentrations in 1ul samples.

  • Olympus MVX Fluorescent Macro scope ( Fluorescence microscope )

    A very sensitive monocular dissecting scope for screening petri dishes of zebrafish embryos. Contains a 0.5NA 2X and a 0.25NA 1X objective. Uses an XCite light source. Lab contains two of these instruments.

  • Sutter P-2000 Laser Based Micropipette Puller ( Micropipette puller )

    CO2-laser based puller for pulling microelectrodes, patch pipetts, or optical fibers.

  • UVP EC3 Imaging System ( Imager )

    One light tight box suitable for imaging gels, chemiluminescent blots, fluorescent small animals, or bacterial colonies.

  • Zeiss LSM 710 NLO two-photon confocal microscope ( Two-photon confocal microscope )

    Confocal and 2-photon fluorescent microscope with environmental chamber for long-term imaging. Has 3 spectrally adjustable channels for confocal/2-photon descanned microscopy. Also contains 3 filter-based channels in both the epi and trans sides for non-descanned 2-photon microscopy. Uses an upright AxioExaminer stand. Contains fixed laser lines at 405nm, 458nm, 488nm, 514nm, 561nm, 594nm, and 633nm and a Coherent Ultra II Ti:Saphire laser tunable from 680nm to 1080nm.

Protocols

  • 20x RapidRun agarose buffer protocol ( Protocol )

    Prepare DNA gel running buffer stock.

  • BAC recombineering protocol ( Protocol )

    How to target your gene of interest while including more regulatory elements.

  • Competent cells protocol ( Protocol )

    General guidelines of competent cells and transformation protocols in lab.

  • Danieau Buffer protocol ( Protocol )

    "This buffer should be used at 1X for raising early zebrafish embryos (0-36hpf) which have been dechorionated."

  • Data processing on Barracuda protocol ( Protocol )

    Useful commands for processing your data (for features not yet incorporated in GoFigure).

  • Data processing on Orchestra protocol ( Protocol )

    Read the "Data processing on Barracuda protocol" first, then proceed to this protocol for guidance on using HMS's computing cluster, Orchestra.

  • DNA loading buffer protocol ( Protocol )

  • Egg water buffer protocol ( Protocol )

    "This buffer should be used for routine collection of zebrafish eggs, raising embryos that still have their chorion on, and for hatched-out larvae. (Danieau buffer should be used for dechorionated embryos)."

  • Glycerol stock protocol ( Protocol )

    Protocol covers making glycerol stock and submitting to the depository. Glycerol stocks provide easy-to-use long-term storage of plasmid in bacteria culture. It is preferable to submit glycerol stocks together when submitting plasmids to the depository.

  • Injection Buffer Protocol ( Protocol )

    10X Injection solution

  • Isothermal Assembly Aliquots Protocol ( Protocol )

    If we ever run out of Isothermal Assembly Reaction Aliquots, here is how to make more.

  • Isothermal Assembly Protocol ( Protocol )

    Isothermal Assembly works by combining a cocktail of exonuclease, polymerase, and ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT overlap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50C.

  • Linker Ligation Protocol ( Protocol )

    Use this procedure to insert small (<100 bps) sections of synthetic DNA into plasmids A linker is made by annealing 2 complementary oligos. The oligos are designed such that once annealed they leave sticky ends which match the sticky ends of the plasmid. This procedure allows complete control of the sequence over small (<100bp) stretches of a plasmid.

  • Mega Mounts Protocol ( Protocol )

    Embryo arrays are agarose mounts made from a plastic template. The plastic template creates "embryo shaped" wells in defined positions in the agarose that allow the embryo to develop in a defined orientation (lateral or dorsal). Embryos can develop normally in the mounts for at least 3 days and be continuously imaged during this time.

  • Methylene Blue Buffer Protocol ( Protocol )

    1000X Methylene Blue Stock. Methylene Blue solution can be added to Egg Water as a fungicide.

  • mRNA Making Protocol ( Protocol )

    Use this procedure to make mRNA for egg injections, kits are available in lab for major steps of RNA making.

  • Nik's modified In-FusionTM Dry-Down PCR Cloning Kit Protocol-at-a-Glance ( Protocol )

    "A modification of the In Fusion PCR Cloning method protocol published by Clontech. Please read the User Manual (PT3754-1) before using this Protocol-at-a-Glance. This abbreviated protocol is provided for your convenience, but is not intended for first-time users."

  • Plasmid Database and Depository Protocols ( Protocol )

    Rules and Protocols regarding lab plasmid Depository.

  • Protocol for Genotyping of FlipTraps by RACE ( Protocol )

    Identifying trapped genes by 3' RACE

  • Simplified DNA Depositing Protocol ( Protocol )

    First time users: Please read the complete protocol before following this simplified procedure.

  • Tricaine Buffer Protocol ( Protocol )

    For anesthetizing zebrafish embryos and adults. The working concentration is 1X to 1.4X (depending on age of tricaine) and should be titrated for each batch.

  • XL1-Blue Cell Transformation Protocol ( Protocol )

    Transformation Protocol for XL1-Blue Subcloning Competent Cells - plasmid transformation protocol modified from Stratagene

  • Zebrafish Bleaching Protocol ( Protocol )

    This protocol should be used to bleach eggs to sterilize them whenever embryos are transfered from the quarantine room to the main fish room. Bleaching only sterilizes the surface of the eggs. It cannot sterilize within the chorion or a lump of fish poo.

  • Zebrafish Egg Collecting Protocol ( Protocol )

    This procedure tells you how to collect eggs for injection, observation, or line maintenance. Zebrafish lay their eggs when the lights come on which is at 9:30am. Thus adults must be setup on the day prior to when eggs are needed (before 10pm when the lights go off).

  • Zebrafish Egg Injection Protocol ( Protocol )

    This protocol describes how to inject zebrafish eggs with RNA, DNA, protein, or small molecules using a Nanoject.

  • Zebrafish Feeding Protocols ( Protocol )

    Recommended feeding times and diets for various stages.

  • Zebrafish Quarantine Protocol ( Protocol )

    Quarantine protocol for importing and transferring fish.

  • Zebrafish round robin crossing protocol ( Protocol )

    In order to restock wild-type fish (AB, TL, Casper, etc.) for common use, a "Round Robin" cross should be completed once a month. To prevent against inbreeding, these steps should be followed.

  • Zebrafish whole mount antibody staining protocol ( Protocol )

    Use this protocol to stain for staining whole embryos with an antibody for fluorescence detection.

Software

  • Cell Flipper ( Software )

    This widget can be used to validate the phase of invidual cells from 3D time-lapse single/multichannel images.

  • Cell Segmentation ( Software )

    Segments nuclei in a 2D confocal image.

  • Cell Tracker ( Software )

    This widget can be used to create tracks of individual cells from 3D time-lapse single/multichannel images.

  • Embryo Counter ( Software )

    This widget can be used to segment 2D circular objects such as embryos in dark-field fluorescent images.

  • GoFigure ( Software )

    A cross-platform image visualization and analysis platform for cell based quantitative analysis. Developed by Arnaud Gelas, Lydie Souhait, Nicolas Rannou and Kishore Mosaliganti in the Megason Lab in the Department of Systems Biology, Harvard Medical School.

  • MegaCapture ( Software )

    MegaCapture is a VisualBasic macro developed by Sean Megason for automating the acquisition of in toto image sets. It is useable on Zeiss microscopes using LSM v4.x software (pre-Zen). MegaCapture can automatically acquire image sets across any combination of dimensions including x, y, z, time, color, x-tile, y-tile, row, and column. Images are exported on the fly and can be compressed allowing very large (100,000 images) image sets to be captured. This software and all sofware from the Megason Lab is licensed under the BSD style open-source license, and as such can be freely used and modified without permission by anyone. We do appreciate attribution (e.g. citation) in resulting papers.


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Last updated: 2011-10-24T15:20:00.432-05:00

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The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016