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Sadly, we had a lot of trouble with our restrictions, and this page will reflect that in including alot of different restriction protocols.

Protocol 1

  1. Pool plasmid and dry on vacuum centrifuge down to about 50uL
  2. Mix the following into one tube:
    1. 4uL Plasmid and RIP
    2. 5uL 10x Buffer
    3. 0,5uL BSA
    4. Fill with water to 47uL (37,5uL)
    5. 1,5uL Enzyme 1
    6. 1,5uL Enzyme 2
  3. Incubate for 2 hours on 37 degress C.
  4. Inactivate the enzymes at 80 degress C for 20 minutes.
  5. Place product on -20 degress C or continue immediately to ligation.

Protocol 2

  1. Fast digest restriction enzymes. Fast digest restriction enzymes have proved more efficient for cutting DNA, and is less time-consuming to work with. Fast Digest enzymes can be bought at Fermentas.
  2. 24 ul water
  3. 2 ul enzyme
  4. 4 ul Fast Digest buffer
  5. 10 ul PCR product
  6. Leave for 15 min at 37 degrees. Afterwards, inactivate the enzyme for 20 min at 80 degrees.

Be aware, only to cut with ONE enzyme at a time.

After cutting with enzyme 1, isolate and purify the DNA fragments on a gel before applying enzyme 2.

The final volume when cutting is 40 ul.

  1. Add 4 ul loading buffer to the eppendorf tube and load directly onto gel.
  2. Run gel and purify from gel.
  3. Eluate with 10 ul when purifying from gel.
  4. Eventually, place your sample in the vacuum centrifuge in order to get a smaller volume and greater concentration before ligation is applied. Final volume prior to ligation should be 5 ul.