Loading RA beads

L. Maves 100703

I. Beads: AG 1-X8 Resin (200-400 mesh Formate Form) from Bio-Rad.

II. All-trans Retinoic acid can be ordered from Sigma. Dissolve in DMSO to make a 10-4M stock. Aliquot to 1 ml and store at ≠80C.

***RA is light sensitive, so whenever working with RA, try to keep overhead lighting to a minimum.***

III. Load beads the day of your experiment (I havenπt experimented with using old beads). Use a blue tip pipette to add a drop of beads to a 1.5 ml tube. Add 100 ul EM to beads to rinse, and pulse spin down the beads-there should be about 25 ul of bead volume. Pipette off EM and rinse again. Spin down beads, pipette off EM, and add 200 ul of RA (10-4 to 10-6). Let beads incubate in RA for no more than 30 min at RT in the dark-keep tubes upright on a flat table shaker. Spin down beads and pipette off RA. Add 200 ul of EM to rinse. Keep beads chilled until ready to use. During implantation, keep beads on ice, protected from light. I turn off overhead lights during implants, with a small lamp on for lighting.

These Bio-Rad beads are about 50 um diameter, but they may swell and have variable sizes after the RA incubation.

EM: Embryo Medium (p/s, f/s): to 1 liter of fresh Embryo Medium, add 20 ml of Sigma penicillin (5000 U)/streptomycin (5mg/ml). Filter-sterilize through a Nalgene 75mm filter unit.