Saccoglossus injection 2005
Injectors: Michelle Roux, Rachael Norris, Mark Terasaki
Quantitative injection method
Fertilization area
Quantitative data:
Size of the oocyte ~360 µm, volume ~23 pl
What was injected this year - the box
chemically synthesized siRNA from Ambion - dissolve in 0.3 ml water. dilute
1:10, this gives 100 pg/ul.
Record of MT injections
Quantitation for picospritzer method
1% injection of 70 kD rh dextran 10 mg/ml was injected by the mercury method
Under the fluorescence dissecting scope, this has approximately the same brightness
as a 1:1000 dilution of the same stock, where a 2 ul drop was put into a 35
mm
dish
with
2 ml silicon
oil
and
pipetted up and down to make small drops
Notes:
We've always had trouble with fertilization. What might work - get a bowl of
sperm from Chris. Fertilize in a glass dish by first putting in 1 squirt
of sea water, then 0.5 squirt sperm, then ~12 oocytes. Walk over to work
bench, start timer. At >1 min, start transferring oocytes to injection
chamber. This 1 minute time period was working with the late season oocytes.
Perhaps
it should be shorter at peak season. With this method, the fertilizations
seemed to be less synchronous, so we did injections straight through (i.e.,
without waiting during the period between 2-3 min). It is possible that the
sensitive period requires very gentle entry of the needle, while afterwards,
it's OK to push the needle in relatively roughly. My experience now is that
I think I can tell when the injection will be unsuccessful by the way that
the needle enters the oocyte - if it snaps in too suddenly, it won't work.
The first time this was done, was listening to "Clair de Lune" by Leon Fleischer,
maybe that put me into the mood.
Room 2
Rachael - Michelle
Etc
Ashtray
