Extraction of DNA from Single Embryos (Sive et al. 2000)
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Information adapted from:
Early Development of Xenopus Laevis: A Laboratory Manual.
2000; (First ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Sive, Hazel (Author), Grainger, Robert M (Author), and Harland, Richard M (Author).
Genomic DNA is used for Southern blotting, for determining gene structure, and for detecting the presence or absence of genes of interest.
Extraction of DNA from Single Embryos
1. Homogenize the embryo using a micropestle in a microcentrifuge tube, containing 0.5 ml of homogenization buffer (see ).
2. Store the homogenate at -20 deg C until ready to process.
3. Thaw the homogenate and add 2.5 ul of proteinase K 920 mg/ml).
4. Mix well and incubate overnight at 55 deg C.
5. Extract with 1 volume of aqueous phenol.
Note: If the DNA isolation is viscous and seems to drag phenol with it, it may be easier to remove the lower phenol layer rather than removing the upper aqueous layer.
6. Extract once with a 1:1 mix of phenol:chloroform, and then once with chloroform.
Note: Embryos appear to contain compounds that inhibit restriction digestion. It is essential to thoroughly extract the DNA in order to remove these inhibitors.
7. Transfer the aqueous phase to a fresh tube and precipitate the DNA by adding ammonium acetate to 2 M and 0.6 volumes of isopropanol.
8. Mix gently but thoroughly by inverting the tube.
Note: The precipitate may be viscous and mixing can take some time.
9. Hold the tube on ice until a stringy DNA precipitate appears (~30 minutes). This may not be apparent from a single embryo before stage 20-30.
Note: If DNA fails to precipitate on ice, store at -20 deg C overnight.
10. Recover the precipitate by centrifuging at 12,000xg for 5 minutes.
11. Wash the pellet in 70% ethanol. Redissolve in 10 ul of TE.
12. Add RNase A to 10 ug/mo and RNase T1 to 10 ug/ml. Incubate for 30 minutes at room temperature.
13. Precipitate the reaction by adding ammonium acetate to 2 M and 0.6 volumes of isopropanol.
14. Centrifuge at 12,000xg for 5 minutes and resuspend the pellet in 20 ul of TE
Note: The older the embryo, the greater number of nuclei and the greater the amount of genomic DNA.