From 2010.igem.org

DNA Double Digestion Protocol

Materials:

􀁺 DNA sample(s) in water or TE buffer 􀁺 10x digestion buffer 􀁺 Restriction enzyme s (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)

Procedure:

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).

Tips:

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!

References:

• Current protocols in molecular biology (3.1.1-3.1.2)