Microinjection
 |
Capabilities:
Location: Room C268 LSRC (668-5308) Cost: $50 per hour |
- Eppendorf FemtoJet/TransferMan NK2 microinjection system
- Installed on an Olympus IX-71 microscope which is also the spinning disk confocal
The microinjection apparatus belongs to the York lab who have generously allowed other people to use the equipment without compensation.
Cost
$50 per hour (money goes to LMCF for personnel costs)
Protocol summary
Grow cells either on 35 mm Matek dishes (buy) or coverslips in standard 35 mm tissue culture dishes. CELLocate coverslips have a grid pattern that allows you to relocate microinjected cells.
Provide own Femtotips and microloaders buy
Solution to be injected must be centrifuged (14 000g for 10 min) and filtered to remove any particulates that may clog the needle. Substance to be injected needs to remain soluble - ie not aggregate, polymerize etc. No glycerol, detergents, azide. Starting concentrations:
- DNA 20-150 ng/µl
- Protein/antibody - 1mg/ml in 1x PBS
Co-injecting marker to identify injected cells - GFP plasmid, dye, fluorescent-dextran etc.
Back fill needle with microloader making sure there are no air bubbles, gently flick thread of needle to move liquid to end of tip. Be careful of dust. Press menu to change needle, attach to system, starting point for settings- pi 150 hPa; ti 0.5 sec; pc 50 hPa.
For more details:
Sample preparation for microinjection