Microinjection

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This information is left here for reference

LMCF microinjection (John York's) Capabilities:
  • Eppendorf FemtoJet/TransferMan NK2 microinjection system
  • Injecting up to 100 pL DNA, antibody, proteins inhibitors etc into adherent cells and some other samples.
  • View cells with phase contrast or fluorescence (green or red)
  • No image capture

 



The microinjection apparatus belongs to the York lab who have generously allowed other people to use the equipment without compensation. John York has been recruited to Vanderbilt and so the microinjection system will be leaving Duke summer 2012.

Detailed specifications

Olympus IX-70 inverted microscope stand

Conventional fluorescence:

  • Green
  • Red

Objectives:

  • 10x/0.3 (Olympus U PlanFl) px= 0.6584 micron
  • 40x/0.6 Ph2 (Olympus LC PlanFl) px= 0.1715 micron
  • 40x/0.9 (Olympus UApo) px= 0.1666 micron
  • 100x/1.35 oil (Olympus U PlanApo) px = 0.0662 micron

 

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) or if you just need a few you can buy them at cost from LMCF

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.