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.
With a Plan 1.0X objective, transmitted light bases, and a micromanipulator controlled Nanoject for microinjections. Lab contains six of these instruments.
8-channel version of the Nanodrop. Capable of measuring DNA/RNA concentrations in 1ul samples.
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.
CO2-laser based puller for pulling microelectrodes, patch pipetts, or optical fibers.
One light tight box suitable for imaging gels, chemiluminescent blots, fluorescent small animals, or bacterial colonies.
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.
Prepare DNA gel running buffer stock.
How to target your gene of interest while including more regulatory elements.
General guidelines of competent cells and transformation protocols in lab.
"This buffer should be used at 1X for raising early zebrafish embryos (0-36hpf) which have been dechorionated."
Useful commands for processing your data (for features not yet incorporated in GoFigure).
Read the "Data processing on Barracuda protocol" first, then proceed to this protocol for guidance on using HMS's computing cluster, Orchestra.
"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)."
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.
10X Injection solution
If we ever run out of Isothermal Assembly Reaction Aliquots, here is how to make more.
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.
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.
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.
1000X Methylene Blue Stock. Methylene Blue solution can be added to Egg Water as a fungicide.
Use this procedure to make mRNA for egg injections, kits are available in lab for major steps of RNA making.
"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."
Rules and Protocols regarding lab plasmid Depository.
Identifying trapped genes by 3' RACE
First time users: Please read the complete protocol before following this simplified procedure.
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.
Transformation Protocol for XL1-Blue Subcloning Competent Cells - plasmid transformation protocol modified from Stratagene
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.
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).
This protocol describes how to inject zebrafish eggs with RNA, DNA, protein, or small molecules using a Nanoject.
Recommended feeding times and diets for various stages.
Quarantine protocol for importing and transferring fish.
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.
Use this protocol to stain for staining whole embryos with an antibody for fluorescence detection.
This widget can be used to validate the phase of invidual cells from 3D time-lapse single/multichannel images.
Segments nuclei in a 2D confocal image.
This widget can be used to create tracks of individual cells from 3D time-lapse single/multichannel images.
This widget can be used to segment 2D circular objects such as embryos in dark-field fluorescent images.
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 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.