- Use kwik-fil capillaries MTW100-4 by World Precision Instruments, Inc.
- Take Petri dish with plasticine.
- Place the capillary in the machine and choose program 70.
- Close the top and press Pull. Wait until it goes back to the initial message screen.
- Take the needle out and put it on the plasticine in the dish. You can start over with new capillary
Loading the needle:
- Prepare your plasmid in the right ratio (centrifuge it first).
- Add a tiny amount (only with the tip of the tip) of fast green.
- Cut the top of a long 20μl eppendorf tip and connect it to a syringe.
- Upload the plasmid and inject it to a needle.
- Break the needle under the binocular using tweezers or tungsten knife.
- Connect positive charged red wire (brown) to the upper electrode.
- Connect negative charged black wire (green) to the bottom electrode (round one). Use masking tape and connect it to the binocular so it will not move.
- Adjust the micromanipulator (the good one…).
- Fill the bottom electrode with Hank's solution all the way to the top.
- Bring the upper electrode into the Hank's solution only to the point it is covered with solution.
- Turn on the elctro-porator and test it: 30V, 25ms, 2 pulses and see if u can see small bubbles (you should…). If you don't see them check your connection and see if there is a big bubble stuck in the bottom electrode.
- Set the configurations: • St.2-3 – 6V, 25ms, 2 pulses (500μs interval) • St.6-8 - 7V-8V, 25ms, 3 pulses (500μs interval)
- Soak the embryo gently (from new-culture) in the Hank's solution in the bottom electrode. Clean it (very jently) if needed.
- Adjust the embryo so it is beside the bottom electrode – you need to see the embryo clearly and to be able to inject comfortably.
- Disassemble the syringe and draw out the piston (always do this when the syringe is disassembled!).
- Connect the syringe, insert the needle to the bottom, inject to the desired position and elevate it slowly until you see green circle accumulate.
- Place the embryo just above the bottom electrode and the upper one precisely above it (see step 5).
- Press Start.
- Every few embryos check if you can see the bubbles. When the Hank's becomes pink replace it.