Isolation of DNA from Red Blood Cells (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.
Isolation of Red Blood Cell Nuclei
1. Anesthetize the frog, and place the animal belly up on a piece of plastic-coated bench paper plastic side up.
2. Introduce a 1-cm^2 opening in the upper ventral thorax to expose the heart (which should still be beating).
Note: Keep the opening small so that the liver does not pop out.
3. Use heavy scissors to cut and remove the shield-shaped bone connecting the shoulders.
Note: The heart should be visible through the silvery pericardium.
4. Expose the heart by carefully lifting and cutting open the pericardium.
5. Inject the heart with 0.5 ml of heparin (2 mg/ml) and allow blood to flow freely from the wound.
6. Collect the blood in a beaker containing 0.85x SSC (see ). Drape the frog over the top of the beaker such that the heart is actually in the solution.
Note: Approximately 3-2 ml of blood will be pumped into the beaker over a period of 10 minutes. If the blood flow stops, a second incision can be made in the heart. Massage the frog's legs to help maintain blood flow.
7. When the procedure is complete wrap the frog in a plastic bag and place overnight at -20 deg C before discarding.
8. Collect the cells by centrifuging in a benchtop centrifuge at about 2000 rpm. Pour off the supernatant, and place the tube on ice.
Note: For the remainder of the protocol, keep the cells on ice and use ice-cold buffers at all times.
9. Partially resuspend the pellet in the residual supernatant and lyse the cells in RSB containing 0.05% nonidet P-40 (NP-40). Start with a small volume (~5 ml), resuspend cells, and then increase volume to approximately 50 ml.
Note: NP-40 is a nonionic detergent that breaks down cell membranes, by not nuclear membranes.
10. Hold on ice for 1 minute and collect the nuclei by centrifuging in a benchtop centrifuge at about 2000 rpm.
11. Pour off the supernatant gently and resuspend the loose pellet by vortexing. When the nuclei are thoroughly resuspended, adjust the volume to 50 ml with RSB containing 0.05% NP-40.
12. Collect the nuclei by centrifuging in a benchtop centrifuge at about 2000 rpm. Repeat steps 11 and 12 until the nuclear pellet is white.
Note: If the nuclei aggregate, dounce gently with a "B" pestle or remove clots by filtering through 2-4 layers of cheesecloth.
13. Store nuclei at -20 deg C in nuclei freezing buffer at a concentration of 1 ng/ml. Proceed with preparation of genomic DNA as described below.
Preparation of Genomic DNA from Red Blood Cells
The preparation of genomic DNA from red blood cell nuclei requires gentle treatment to avoid shearing the DNA. Avoid vortexing and violent phenol extraction where possible. the following procedure should yield several milligrams of DNA, more than 200 kb in size, suitable for cosmic cloning and genomic Southern blots.
1. Thaw nuclei prepared in the protocol above, and transfer to a 50-ml tube (Corning). Dilute to 100-200 ug/ml in RSB and add proteinase K to a final concentration of 200 ug/ml. Mix gently - do not vortex.
2. Add one volume of 0.6 M NaCl, 20 mM Tris-HCl (pH 7.4), 20 mM EDTA, and 1% SDS and mix gently, but thoroughly.
Note: The solution will become very viscous as the nuclei lyse.
3. Precipitate the DNA by adding 0.25 volumes of 10 M ammonium acetate and mixing thoroughly.
Note: The DNA should precipitate immediately.
4. Use a thin glass rod, or a heat-sealed pasteur pipette, to spool the DNA out of the mixture. Discard the remaining solution and replace with 20 ml of 70% ethanol. Use a second pasteur pipette to return the spooled DNA to the tube.
5. Wash the DNA by gently inverting the tube, until it is completely white and rather stringy looking.
6. Carefully draw off the ethanol with a pasteur pipette, avoiding the DNA.
7. Resuspend the DNA at an estimated concentration of 200 ug/ml.