Albumin–Agarose
MATERIALS
Reagents
30 mL thin albumin (from unincubated eggs)
30 mL simple saline (0.72% NaCl), autoclaved
0.18 g Bacto agar (BD 214010)
30 µL penicillin (10,000 U/mL)/streptomycin (10 mg/mL) (Sigma-Aldrich)
Equipment
Conical vial (50 mL), sterile
Flask (100 mL)
Hot plate/stirrer
Imaging and storage dishes of choice: two-well cover glass chambers (for in vitro inverted imaging) or 35-mm Petri dishes (for intermediate in vitro embryo storage)
Water bath preset to 49°C
METHOD
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This procedure was derived from Darnell and Schoenwolf (2000).
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1. Using a hot plate/stirrer, add simple saline to a 100-mL flask and bring it to a boil.
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2. Add agar and stir until dissolved.
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3. As the agar dissolves, collect thin albumin in a sterile 50-mL conical vial and set in a 49°C water bath.
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4. Once the agar has dissolved, equilibrate to 49°C in the water bath.
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5. Set out the imaging and storage dishes of choice. Remove all lids.
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6. Add the warm albumin to the agarose and swirl to mix. Add penicillin/streptomycin to the mixture (to a final concentration of 5 U/mL penicillin).
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7. Using a sterile pipette, quickly add the appropriate amount (e.g., 450 µL per chamber of a two-well cover glass) of albumin/agarose mixture to each chamber. Be careful not to introduce bubbles.
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8. Replace lids on dishes and chambers and allow them to dry at least 1 h, but preferably overnight. (Plates are good up to 1 mo after pouring.)
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9. Dispose of unused albumin–agarose.
- © 2011 Cold Spring Harbor Laboratory Press
