1.Obtain 65-100 µl of blood by retro-orbital bleed with a heparinized microcapillary tube. Expel blood immediately into a 1.5 ml microfuge tube containing 20 µl of 10 mM EDTA. Mix immediately to prevent clot formation. Store on ice until processing.
2.Add 200 µl Lysis Buffer to each tube and vortex to suspend evenly.
3.Microfuge 25 seconds at 16000xg to pellet nuclei.
4.Remove and discard supernatant and repeat steps 2-4 two more times, or until no hemoglobin remains.
5.Resuspend nuclear pellet in 100 µl PBND with 60 µg/ml proteinase K and incubate at 55 ℃ for 60 minutes (or overnight, if convenient).
6.Heat samples to 97 ℃ for 10 minutes to inactivate proteinase K.
7.Add 1-5 µl of DNA solution for a 25 µl reaction.
Reagents:
1) Lysis Buffer
0.32 M Sucrose
10mM Tris-HCl (pH 7.5)
5 mM MgCl2
1% v/v Triton X-100
2) PBND ( Buffer with Nonionic Detergents)*
50 mM KCl
10 mM Tris-HCl (pH 8.3)
2.5 mM MgCl2
0.1 mg/ml gelatin
0.45% (v/v) Nonidet P40
0.45% (v/v) Tween 20
Autoclave to sterilize and dissolve gelatin
Store frozen
*Add proteinase K (60 µg/ml) immediately prior to use)
Typical 25 µl reaction:
1-5 µl DNA
2.5 µl 10x Perkin Elmer buffer, 1.5 mM MgCl2 (final)
2 µl 2.5 mM dNTP mixture (2.5 mM each dNTP, 200 µM final)
0.5 µl 20 µM forward primer (0.4 µM final)
0.5 µl 20 µM reverse primer (0.4 µM final)
0.1 µl Taq DNA polymerase (can decrease to 0.05 µl)
dH2O to 25 µl
