| Formamide
|
50% |
| 20X SSC (pH 4.5)
|
5X |
| Triton X-100
|
0.25% |
| SDS
|
1% |
| Salmon sperm DNA |
100 μg/mL |
|
| Prepare a 10 mg/mL stock solution of salmon sperm DNA in H2O. Autoclave for 20 min to shear and denature the DNA. Store at 4°C or in aliquots at -20°C.
|
| Prepare 40 mL of SDS hybridization buffer in H2O and measure the pH. If the pH is not between 5 and 6, check the starting solutions and remake. A slightly acidic pH is essential
to prevent the embryos from disintegrating.
|
| Important: Just before preparing hyb buffer, remove a working volume of 20X SSC and use concentrated HCl to adjust its pH
to 4.5.
|
| Take precautions to ensure that the hyb buffer is RNase-free. |
| Hyb buffer can be stored at -20°C but should be warmed before use in order to resuspend the SDS. It can also be made fresh
and kept at 65°C for the duration of the procedure.
|
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