Mouse tissue lysis for genotyping protocol

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Solutions/reagents:

Equipment:

  • Incubator
  • Centrifuge

Steps:

  1. Tissue lysis to release DNA
    Measure out 100 µl of Taq DNA polymerase 10X buffer with MgCl2 into tail or tissue chunk.
    Add 2 µl of proteinase K.
  2. Incubate at 50°C for 3 - 12 hrs(overnight) with shaking at 300 rpm.
    proteinase K is stable over a broad pH range (4.0 to 12.5, optimum pH 8.0) and is also stable over the temperature range of 25 to 65°C.
  3. Proteinase K inactivation
    Incubate at 95°C for 10 - 20 mins with shaking at 300 rpm.
    Centrifuge at a speed of 13000 rpm for 3 mins at room temperature and aspirate out 80 µl of top layer into sterile 1.5-ml microcentrifuge tube (2).
    Discard bottom layer.
  4. Storage before PCR
    NOTE: If sample is/are used within 1 hr, set temperature to 4°C and use, else store it at -20°C.

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