Isolation of Genomic Dna from Mouse Tail or Spleen
(after Laird et al., 1991)
Overview
• Lysis (steps 1-2)
• Isopropanol precipitation (3-4)
• Transfer of precipitate (5-6)
1. Cut 1/2 to 1 cm of tail from mice 1 to 2 weeks old, or cut 1/3 from spleen taken during perfusion, and place into a 2 ml labeled eppie tube.
2. Add 1 ml of lysis buff to each tube. Incubate overnight in shaking bath at 55 C. 25 ml of the lysis buffer is enough for two 96 well plates. The lysis buffer is made from:
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Stock Solution
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Concentration
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To make 25 ml
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|
1 M Tris-HCl pH 8-8.5
|
100 mM
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2.5 ml
|
|
500 mM EDTA pH 8
|
5 mM
|
0.25 ml
|
|
20% SDS
|
0.2 %
|
0.25 ml
|
|
5 M NaCl
|
200 mM
|
1.0 ml
|
|
water
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…
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20.7 ml
|
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*Proteinase K (20mg/ml)
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250 µg/ml
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1.0 ml
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* store at room temperature and add just prior to use
3. Spin tubes for 5 minutes at 10-13K/min and extract supernatant into a clean tube.
4. Add 1 volume of isopropanol to each tube containing lysate and mix -- do NOT vortex. DNA should precipitate.
5. Burn the ends of glass pipets to form a small hook (one labeled pipet per animal) to fish out the DNA mass. The DNA should stick to the glass hook. Remove and dip into a tube of 70% EtOH (one labeled tube per animal) to wash the pellet of DNA. Remove and invert the pipet and allow pellet to dry for at least 10 minutes.
6. Place dry pellet into a new tube with 200 µl dionized filtered water. Agitate if necessary to remove pellet from pipet. Heat at 55 C for 30 min to 2 hrs to dissolve if necessary. Store at 4 C. Test concentration of this solution by dissolving 2 µl of DNA solute in 200 µl TE (Tris-EDTA) or deionized filtered water. We use an inexpensive Gene Quant II RNA/DNA Calculator. For longterm storage of DNA save in TE solution.
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