The lab in which I work has recently switched from injecting
dessicated dechorionated embryos using needles whose tips are made by
breaking against a cover slip to injecting undessicated embryos
directly through the chorion using pre-pulled Eppendorf Femtotip
needles. The Femtotip needles work great in that they pierce the
chorion very easily. However, I haven't gotten any transformants yet
and I suspect that I'm not injecting enough DNA. With the hand-peeled
embryos under halocarbon oil, it was readily apparent that liquid was
going in. It is very difficult for me to tell if I'm actually getting
DNA in through the chorion though.
I am using a synthesis of various suggested protocols. I flood the
egg collection plate with ethanol so they are immersed for 15 minutes
(this seems to make the chorion easier to penetrate), then transfer
them to an agar coated cover slip. They stick well to the agar, so I
can avoid using double-stick tape. I line them up and let the ethanol
evaporate, then cover them with halocarbon oil to help make the chorion
transparent (sort of). The pressure for injection comes from a 50 ml
plastic syringe connected to the
Femtotip with plastic tubing. I started putting Schilling green food
coloring into the injection buffer (at 5% final concentration) to
visualize the liquid being injected. I sometimes can barely see green
being injected, but usually I can't tell. I put the cover slip with the
embryos onto a small food plate in a humidified chamber at 17 degrees
C. When they hatch 2 days later, I don't see any green in the larvae.
My questions for those of you using Femtotips are:
1. How long do you immerse the eggs in ethanol?
2. Do you use oil to make the chorion transparent?
3. How do you provide pressure to the tip?
4. What temperature do you put the embryos at after injection?
5. How do you know whether anything is going into the embryo?
6. If you use dye, can you see anything after injection?
7. What concentration of wings-clipped helper do you use?
Thanks for your advice!