Partial Restriction Digest

  1. Add the following to a 1.5 mL tube:

    Reagent Amount
    Plasmid DNA 1 μg
    10X Buffer 2 μL
    Enzyme 1 1 μL
    Water To 20 μL
  2. Incubate at the recommended temperature for 1 hour (or to completion).

  3. To the completed digest, add:

    Reagent Amount
    10X Buffer 8 μL
    Water 72 μL
  4. Label the tube “A” and place on ice.

  5. Aliquot 20 μL from A to tubes labeled “B,” “C,” “D” and 10 μL to a tube labeled “E." “A” should have 30 μL remaining. Keep all tubes on ice.

  6. 1 μL of Enzyme 2 to “A”. Mix well.

  7. Transfer 10 μL from “A” to “B.” Mix well.

  8. Transfer 10 μL from “B” to “C”, then 10 μL from “C” to “D”, and 10 μL from “D” to “E.” Mix well each time, and make sure to change tips in between each transfer.

  9. Incubate all tubes at the recommended temperature for 30 s. - 4 min. (first try with a 2 min incubation). Place on ice.

  10. Add the first buffer from the PCR Purification kit to each tube, then pool all the tubes. Perform the rest of the PCR purification.