Embryo Injections

Pre-injection prep

2.5% Ficol in 1/3X MR (autoclaved)

1/3X MR

Testes (less than a week old in 1X MR (4° C)

Needles

RNA (on ice)

Fill metal trays with ice and put on metal cookie sheets

Squeezing frogs: get frogs + an extra bucket w/ salt water.

have a glass Petri dish lid for each frog with a little bit of ddH20 in each

squeeze eggs into dishes

shred testes (need 1/4 testes for 6 plates) with forceps in 1X MR

Distribute sperm between plates w/ eggs

Add H20 to eggs/sperm plates and mix with transfer pipette

= Eggs will fertilize in ~20 minutes =

Make cysteine (fresh for day of use) 2.5% in H2O (2.5g in 100 ml) pH 8.0 with NaOH

Fill grided plates with 2.5% Ficol in 1/3 MR, place on cookie sheet

After embryos turn, pour off H2O, add 2.5% Cysteine pH 8.0, stir to mix

~5 min later check to see if embryos are de-jellied (keep Cysteine to a minimum)

Rinse de-jellied embryos well:

pour off cysteine

wash 2X with H2O

wash 2-3X with 1/3 MR

Aliquot embryos between gridded plates (20-40 embryos/plate)

Inject

Needle Prep

place RNA into needle ~5mm of liquid in capillary tube
cut needle tip so that it makes a bubble that measures 12 increments at highest scope power; this will represent 10 nl of RNA