DNA prep for SNP TaqMan assay

Protocol A (Fast approach)

1.Obtain 2 mm of the tail and place directly into 75 uL alkaline lyse buffer (NaOH [25 mM],Na₂-EDTA [0.2 mM], pH=12) in a PCR tube.

2.Samples heated at 95ºC for 10 min to 1 h. After heating, samples are cooled at 4ºC and neutralized with 75uL of Tris-HCl [40 mM, pH=5].

3. Centrifuge for 5 min at 4ºC 12000 rpm , and 1 to 5uL of the supernatant is used per each PCR reaction.

Protocol B

  Reagent:
   Digestion Buffer (Pure H₂O 415mL,Tris-HCl [1M] pH 8.0 50mL, NaCl [5M] 20mL,  SDS 10% 10mL and EDTA [0.5M] 5mL; 500mL in total)

1.Cut the last 2 mm embryonic tail and place directly into an 1.5mL tube.

2.Add 200 uL of Digestion Buffer and 2uL of Proteinase K (10mg/mL).

3.Boil at 55 ºC.

4.Centrifuge at 12000 rpm for 15 min.

5.Transfer the supernatant to a new 1.5mL tube.

6.Add about 180 μL of isopropanol (volume 0.7).

7.Mix well by vortexing gently until you see the DNA precipitate. Centrifuge at 14000 rpm for 25 min at 4ºC.

8. Discard the supernatant, and add 100uL 70% pre-chilled EtOH. Repeat centrifuge at 14000 rpm for 5 min, and aspirate the EtOH.

9.Resuspend DNA with 100uL H₂O, and let dissolve at 37ºC for 1 hour.

10.Save your DNA extract at 4 ºC until use.

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