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 PCR 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 (PCR 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 PCR 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