Injection Mixture Preparation For Egg Microinjections NADINE NASSIF
Injection Buffer:
| 1X | final concentration | mix |
|---|---|---|
| Na Phosphate (pH~6.8-7.8) | 0.1 mM | 0.01 ml of 100 mM |
| KCl | 5 mM | 0.1 ml of 500 mM |
H2O to 10 ml, filter sterilize
to prepare Na Phosphate: make 0.1 M solutions of both monobasic
and dibasic. pH one solution with the other to pH~6.8-7.8. Use
this solution in the mix.
Injection Mixture:
5 ul of 2X injection buffer
4 ul of DNA (0.5 - 1.0 mg/ml, in H2O)
1 ul green food coloring dye
(optional: 1 ul of carbenicillin at 1 mg/ml)
Notes:
Can also resuspend DNA in 1X injection buffer to inject
Do not resuspend DNA in TE(10+1); the EDTA concentration is too high and can kill the embryo. If you want, you can resuspend in TE(10+0.1); otherwise, just resuspend in H2O.
The carbenicillin is an optional step that you can try if you find that a large number of your injected eggs are turning brown and dying shortly (~24 hrs) after injection. The drug doesn't help transformation frequency at all, and we don't know for a fact if it helps the embryo survive any longer. But it seems to help prevent the embryos from turning brown so quickly, and it hasn't hurt survival or transformation frequencies.
Injecting
Let the females lay eggs on freshly yeasted food for at least an hour to get rid of the older eggs; transfer them to fresh food once again during this hour.
Let the females lay eggs for ~20 minutes on freshly yeasted food. Pick ~35-40 eggs to inject, line them up on double-sided tape on the edge of a slide. We don't dechorionate them. Overlay with enough halocarbon oil to cover the eggs, and inject. When you're finished injecting that batch, peel up the tape and remove quite a bit of the halocarbon oil; eggs left with too much oil on them tend to smother and die. Put the tape with the eggs on freshly yeasted food, place in a moist environment (we put them in a tupperware container with moist towels and incubate at 25° C). Survivors should emerge within 24 - 48 hours.
