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DNA Double Digestion Protocol


  • DNA sample(s) in water or TE buffer
  • 10x digestion buffer
  • Restriction enzymes (EcoRI or SpeI or XbaI or PstI)
  • DNA loading buffer (if electrophoresis is subsequent)
  • Agarose gel 1.5% (or different depending on expected band sizes)


1. Test the concentration of the DNA sample(s).

2. Pipet the following into a microfuge tube:

20uL reaction system     50uL reaction system

DNA around 1ug     around 2.5ug

10x Digestion buffer 2uL     5uL

1st Enzyme 1-1.5uL     2.5-4uL

2ndEnzyme 1-1.5uL     2.5-4uL

ddWater Rest of volume Rest of volume

3. Incubate at recommended temperature (37.0 degrees) for 2 or 4 hours (0.5~2hfor enzymes of NEB, 4h for enzymes of Takara).

4. Take 2 to 5 uLof the digested sample, add loading buffer, and run it on the agarose gel to check the result, or take the entire sample to run to extract a wanted fragment).


1. DNA:

  • For identification of DNA, use 0.4 ug/uL DNA; (or 2uL from a nice DNA mini prep)
  • For cloning, 1ug/uL DNA is enough.

2. Buffer: we’d better use the buffer that comes with the enzyme, which means buffers from other company may cause some abnormal results.

3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the total reaction volume (example: 2 uL for 20 uL reaction system). If you want to do overnight digestion, add less enzyme (example: 1 uL for 20 uL reaction system). It is necessary to point that too many enzymes will reduce the efficiency of enzyme digestion with glycerol in it.

4. Gel: make sure to run the uncut DNA as a control along with the digested DNA sample(s). And, always run a DNA marker!


  • Current protocols in molecular biology (3.1.1-3.1.2)

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