Overview
This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
—–EcoRI–XbaI–Part–SpeI–PstI—–
See the biobrick assembly schedule for more information on using this technique.
Materials
- Prepared DNA from miniprep, PCR, or Gel Extraction
- Restriction Endonucleases
- With corresponding 10X buffer. NEBuffer 2 can be used for most applications.
- BSA
- Antarctic Phosphatase
- Distilled water
Procedure
1. Quickly vortex all ingredients (Buffer, BSA, DNA) before beginning. 2. Add the following in a micro-centrifuge tube:
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- 5μl of Buffer (usually NEBuffer 2);
- 1μl of BSA;
- 0.5 picomoles DNA normally uses 10μL of miniprep or 5μL of purified PCR product.
- Water to make 48μl.
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3. Vortex Enzymes and add 1μl of each to the tube.
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- If you’re digesting purified PCR product (i.e. “insert”), add 1μl of DpnI to the reaction.
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4. Incubate reaction in a 37°C water bath for at least one hour.
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- If your digesting a “vector”, add 1μl Antarctic Phosphatase and 6μl of Phosphatase buffer after 2 hours of incubation and incubate for another hour.
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5. Heat kill the digests for 20 minutes at 80°C. 6. Store digested DNA in the refrigerator (4°C)for use in the very near future.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- DpnI eliminates the background from your PCR template
- Antarctic Phosphatase eliminates background from the vector self ligating.
- If you’re not getting good digestion it might be because your enzyme is bad. Double the digestion time and see.
- Longer digest gives more complete digestion, especially if you have >1µg of DNA, but can sometimes give nonspecific digestion
- Beware the the NEB double digest chart. For EcoRI and PstI double digest it recommends using the EcoRI NEBuffer. However based on my digests both NEBuffer 2 and 3 work better; with NEBuffer 2 giving the most complete double digestion. It is funny that they recommend the EcoRI buffer because their chart also says that PstI is only 50% active in that buffer.