Genotyping protocol

  1. cut the tail for about 0.5~1cm.
  2. add 200µl Direct PCR lysis buffer and 10 µl proteinase K (20mg/ml, -20 oC).
  3. incubate at 65 oC overnight.
  4. heat samples at 85 oC for 45 min to inactivate proteinase K.
  5. genotyping PCR.
     

    (µl)

    5 ×Gotaq PCR Buffer

    4

    MgCl2 (50mM)

    0.8

    dNTPs (10mM)

    0.5

    Primer Mix ( 10µM)

    1

    Tail DNA

    2

    Gotaq polymerase

    0.2

    ddH2O

    11.5

    Total

    20

    Step 1: 95oC, 4min
    Step 2: 95oC, 30 sec
    Step 3: 55 or 60 oC, 30 sec
    Step 4: 72oC, 1 min
    Step 5: Repeat steps 2-4 for 40 cycles
    Step 6: 72oC,10 min.

  6. Run 2% or 3% gel.