DNA double digestion protocol
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Materials
- 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 0.8% (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
|
| 2nd Enzyme | 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 (2h for enzymes of NEB, 4h for enzymes of Takara).
4. Take 2 to 5 uL of 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).
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)
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