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  • Protocol to purify PCR products in preparation for cloning using Proteinase K (P8107)

    1. Pool up to 400 ul of PCRs, containing 1 ug of the desired product. (If nonspecific products are present in a noticeable amount, gel purify purification is recommended.)
    2. Add 1 uL (0.8 U, ~20 ug) Proteinase K. (Proteinase K is active in most common buffers including common PCR buffers like Standard Taq, Q5, and OneTaq).
    3. Incubate for 60 min at 37°C.
    4. Optional: Inactivate the Proteinase K reaction mixture for 20 min at 75°C; Enzyme is completely inactivated only at 95°C for 10.
    5. Extract once with phenol:chloroform and once with chloroform.
    6. Add 0.2 vol 10 M ammonium acetate and 2.5 volumes of ethanol.
    7. Mix well and store it for 30 min at 4°C.
    8. Recover the precipitated DNA by centrifugation at maximum speed for 5 min at 4°C in a microfuge.
    9. Discard the supernatant and then wash the pellet with 70% ethanol.
    10. Centrifuge the solution again, remove supernatant, and then allow the pellet to DNA to dry in the air. These steps will remove free nucleotides but not small oligos/primers.
    11. Dissolve DNA pellet in TE (pH 8.0).