マウスの尻尾からのDNA調製のプロトコールです。
↓ Tail(1.5-2 cm)in microcentrifuge tube (Assist No. 72.693, 2ml tube)
↓ Add 1.2 ml of Lysis Buffer
↓ Add 30 mcl of 10mg/ml Protenase K
↓ Incubate at 50℃ for 6hr〜 overnight and mix every 1〜 2 hr
↓ Spin for 1 min at 15,000 rpm by microcentrifuge
↓ Transfer 1 ml of lysate to new tube (Nunc No.374632, 15 ml tube)
↓ Add 600 mcl of Extraction buffer and mix
↓ Add 2.4 ml of 7.5 M NaI and mix
↓ Incubate at 60℃ for 10 min
↓ Add equal volume (4 ml) of isopropanol
↓ Gentry mix the contents by inverting the tube
↓ Transfer the DNA aggregate to Eppendorf tube
with Pipetman P-1000 (200〜 300 mcl)
↓ Add 40 % isopropanol to a final 1ml and vortex
↓ Spin for 1min at 15,000 rpm by microcentrifuge
↓ Remove sup.
↓ Add 1 ml of 40 % isopropanol
↓ Spin for 1 min at 15,000 rpm by microcentrifuge
↓ Remove sup.
↓ Rince with 70 % ethanol
↓ Dry up the DNA pellet for 2〜 3 min with vacuum centrifuge
(Don't completely dry!)
↓ Dissolve the DNA pellet in 100 mcl of TE
↓ Heat at 65℃ for 15〜 30 min
↓ Spin for 1min at 15,000 rpm by microcentrifuge
↓ Final DNA concentration is 300〜 500 ng/mcl
Lysis Buffer final conc.
1 M Tris-HCl (pH8.0) 10 ml 50 mM
5 M NaCl 4 ml 100 mM
0.5 M EDTA 8 ml 20 mM
10 % SDS 20 ml 1 %
D.W. 158 ml
Extraction Buffer
130 mM Tris-HCl (pH 8.0)
65 mM EDTA
2.5 % Sodium N-lauroylsarcosine
Ref.
L Wang, K Hirayasu, M Ishizawa, and Y Kobayashi
Purification of genomic DNA from human whole blood
by isopropanol- fractionation with concentrated Nal and SDS.
Nucleic Acids Res. 1994 22: 1774-1775. |
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