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Add the following reagents into a round-lidded PCR tubes:

PCR insert (µL)
Plasmid (µL)
X (~3-5 µg)
FD 10x buffer
Restriction enzyme
up to 20 µL
  1. Mix tube(s) gently by tapping.
  2. Centrifuge for 1 minute at 4,500 xg.
  3. Incubate tube(s) for 2 hours at 37°C.
  4. Heat inactivate at 65°C (or 80°C, depending on the restriction enzyme(s) used) for 20 minutes.The heat inactivation temperature for the restriction enzymes can be found in the protocol folder or online, and for double digests, the larger of the two heat inactivation temperatures should be used.
  5. Cool tube(s) on ice for 2 minutes.
  6. Add 1 µL of Calf Intestinal Phosphatase (CIP) and X µL of 10x NEB buffer to the plasmid tube. Note: CIP is not necessary for double digests, and is only necessary when planning on following the digest with a ligation.
  7. Incubate the plasmid tube at 37°C for 1 hour. The tube with the insert may remain on ice.
  8. Run both the plasmid and insert digests out on an agarose gel. Note: to reduce the risk of cross-contamination, ensure that there is ample space between the samples.
  9. After the gel has run, cut out the desired bands using UV light and place the bands into new and separate microcentrifuge tubes.
  10. Purify the samples using the Geneclean II kit. Note: If the amount of water in the glassmilk container is low, add enough ddH2O that the ratio of liquid to solid is approximately 1:1. Also, in step 12 of protocol 5.1 (purifying from solutions) and step 18 of protocol 5.2 (purifying from agarose gel) use 13 µL of ddH2O to extract the DNA.
  11. Quanitfy DNA samples. Note: a good yield is ~30-150 ng/µL.