embryo collection
- collect embryos on apple juice plates, e. g. 0-4h
- cover embryos on the plate with Klorix and incubate for one
to two minutes to remove the chorion
- pour the Klorix with the embryos through a net to collect
the embryos
- wash the plate with water and pour the water with embryos
through the net
- repeat washing with water for several times
- wash the collected embryos in the met with water, dip on a
paper towel to remove excess liquid
- transfer embryos with a small brush into an Eppi tube
whose weight was noted before
- determine weight of the embryos, briefly spin to collect
embryos on the bottom, 1 mg corresponds to 100 Embryos (ref. Ashburner)
- freeze in liquid nitrogen and store the tube at -80ºC
total extract:
- add 2xLaemmli buffer (optional with 8M urea), 10 µl per 1
mg/100 embryos
- sonicate, e. g. 3 x 30 sec.
- boil 5 min, 100ºC
- spin 5min
- store in aliquots at -80ºC
- load 3 µl (=30 embryos) per lane
soluable and nuclear extract
- transfer frozen embryo pellet or living embryos into a
Dounce homogeniser
- add 50 µl lysis buffer per 1 mg/100 embryos
- briefly spin the crude lysat
- extract with an equal volume of trifluortriclhorethan to
remove lipids
- spin 15 min, 14k, 4ºC
- alternatively spin the extract in the UZ
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