Kuwabara Lab – Modified for Herman Lab
I. Making agarose injection pads (needs to be done at least one day before!)
Lay cover slips (22x50mm) on clean surface, making sure edges do not overlap. Many cover slips can be made in advance and stored in the original box.
Use 2% agarose (in the freezer). Melt in heat block at about 100°C, then turn heat down to about 70°C (keeping it at high temperatures will burn the agarose).
While hot, drop small drop of agarose onto cover slip with a glass Pasteur pipet, then quickly place another cover slip over it to spread it out. Leave second cover slip on until dry (less than a minute).
Allow pads to desiccate overnight in an open box or on bench.
II. Preparing needles
Have ready a small freezer box or CD case with a strip of modeling clay inside for holding the needles.
Use the Narishige needle puller to make needles. We use Kwik-Fil Borosilicate Glass Capillaries (1B100F-4 World Precision Instruments). The settings on the needle puller should not be changed unless absolutely necessary.
To load needles; use 0.8-1.10x100mm glass capillary tubes. Flame center until glass softens, remove from heat and quickly pull to generate a needle that is fine enough to fit within the back-end of injection needle. Back fill injection needle using the capillary tube loaded with 1-2ul injection solution. Bubbles may form – shake the needle gently or just wait for them to float to the top. Do not inject with bubbles in the needle!
Needles load very quickly, so no need to fill more than 2 at a time. Solutions will evaporate fairly quickly.
III. Injection Solutions
DNA: Prepare 10ul of injection mix. Generally, inject 20ng/ul of test DNA plus an appropriate concentration of marker (see below):
- unc-119 marker: 40ng/ul; inject unc-119 worms, pick WT progeny.
- rol-6 marker (pRF4): 100ng/ul; inject any genotype worms, pick rol-6 progeny.
- str-1::GFP marker: 40ng/ul; inject any genotype worms, pick progeny with GFP in head (one bright spot).
- SUR-5::GFP: 100ng/ul; inject any genotype worms, pick progeny spotted with GFP through whole body.
Markers are used as a control to ensure that the injection was successful.
Dilute test DNA and marker in ddH2O.
If test DNA does not yield any correct progeny at 20ng/ul, try 10ng/ul. If this doesn’t work, try 50ng/ul.
RNA: Generally inject 1ug/ul solution.
IV. Preparing to Inject
Be sure that you have the following:
Agarose pads
Heavy mineral oil
Needles
DNA/RNA solution
M9 buffer
Seeded NGM plates (1 plate/injection)
Worms (young adults, not starved or desiccated)
Back load needles with DNA/RNA solution. Allow time to fill.
Mount filled needle on microinjector arm. Center and focus needle using low power objective, so tip is in center of field. Raise needle, turn on air to provide forward pressure.
Add drop of oil to agarose pad and place a small piece of pulled capillary tube in the center of the oil. This will be used to break off the tip of the needle so that solution can flow out.
Be sure that the capillary is slightly off center, and then lower the needle down into the oil.
When needle is focused, move stage so that the capillary is near the needle, then move to 40x objective.
Rub needle against capillary so the tip of the needle breaks at an angle. Then check the flow of the solution by depressing the foot pedal.
Beveling will help to ensure good flow rate during injection.
Raise needle and change objective back to low power.
V. Mounting worms on agarose pad
Place drop of oil on pad, place pad on inverted 15mm plate lid.
Select young adult worm and move it to portion of plate without E. coli. Then, pick up worm with pick.
Allow worm to thrash on pick until enough of its body is visible so that it can be made to stick to pad. Guide worm onto pad, so that it lies down as straight as possible. Mount as many worms on a single pad as you feel capable of injecting in a short time (before worms desiccate. Three is a good number).
Quickly move pad to microscope stage and align body position. Move to high power objective.
Once worm is at desired angle, slowly lower needle, but keep away from worm.
Focus germline distal core region (gonad – has a honeycomb-like appearance and is on the side opposite of eggs) and tip of needle in the same focal plane. This is very important!
Using the stage, bring worm to the needle, until it is making a depression into the worm, about a third of the way into the worm. GENTLY tap the edge of the micromanipulator until the needle breaks through the cuticle and is clearly in the gonad. Depress the foot pedal – should see germline fill with liquid.
After completing injection of gonad arm, attempt to inject other arm unless worm is desiccating.
Repeat with any other worms on pad.
Raise needle. Move pad to dissecting scope. Add M9 buffer dropwise to rehydrate worm(s) on pad. Once worm is thrashing around or is loose from the oil, use a Pasteur pipet to suck up the worm and place it on a seeded NGM plate.