General Lab
Embryo culture plates
- IVF D_pealeii embryos are cultured directly on 0.2% Agarose in sea water (or flasks, or dishes, etc)
| Ingredient | Quantity | Final Concentration |
|---|---|---|
| Agarose | 0.2g | 0.2% |
| Sea water | 100mL | - |
| Total | 100mL |
Ca-Mg2 Free Artificial Seawater (ASW)
| Reagent | Quantity (for 500 mL) | Final concentration |
|---|---|---|
| NaCl | 13.1g | 450 mM |
| KCl | 0.335g | 9 mM |
| Na2SO4 | 2.31g | 30 mM |
| NaHCO3 | 0.105g | 2.5 mM |
| Tris-HCl | 5mL 1M (pH 8.0) | 10 mM |
| EDTA | 2.5mL of 0.5M (pH 8.0) | 2.5 mM |
| HEPES | 12.5mL of 1M (pH 6.9) | 25 mM |
| Nuc Free Water to 500mL |
- Filter sterilize and store at 4°C
- pH 6.9-7.4
Artificial seawater (ASW)
High salt PBS (for better DAPI penetration)
| Reagent | Quantity (for 500 mL) | Final concentration |
|---|---|---|
| NaCl | 1.06g | 500 mM (363mM added to 137mM in std PBS) |
| PBS 1x | to 50 mL |
Clearing
Detergent Mix (1 L) (from Jenny McCarthy)
| Reagent | Quantity (for 1L) | Final concentration | Notes |
|---|---|---|---|
| 10% SDS | 100mL | 1% | (may be filtered) |
| Deoxycholate | 5g | 0.5% | (wear a mask when handling the powder) |
| 1 M Tris-HCl (pH 8.0) | 50mL | 50 mM Tris-HCl (pH 7.5) | |
| 0.5M EDTA (pH 8.0) | 2 mL | 1 mM EDTA (pH 8.0) | |
| 5M NaCl | 30 mL | 150 mM NaCl |
Clearing Solution-1.1
- 8 to 10% (v/v) THEED (Sigma-Aldrich, 87600-100ML)
- 5% (v/v) Triton X-100 (Roth, 3051.2)
- 5% (w/v) urea (Roth, X999.2) in dH2O