Richard Lab:Restriction Digest
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This protocol is for the typical ligation of DNA done using the Richard Lab New Standard Assembly.The consensus digestion protocol should be consulted if deviating from the procedure outlined here.
This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
See the biobrick assembly schedule for more information on using this technique.
- Prepared DNA from miniprep, PCR, or Gel Extraction
- Restriction Endonucleases
- With corresponding 10X buffer. NEBuffer 2 can be used for most applications but check this chart to be sure.
- Antarctic Phosphatase
- Distilled water
- Quickly vortex all ingredients (Buffer, BSA, DNA) before beginning.
- Add the following in a micro-centrifuge tube:
- 5μl of Buffer (usually NEBuffer 2);
- 1μl of BSA;
- Water to make 48μl.
- Vortex Enzymes and add 1μl of each to the tube.
- If you're digesting purified PCR product (i.e. "insert"), add 1μl of DpnI to the reaction.
- Incubate reaction in a 37°C water bath for at least one hour.
- If your digesting a "vector", add 1μl Antarctic Phosphatase and 6μl of Phosphatase buffer after 2 hours of incubation and incubate for another hour.
- See the Bio-Brick Assembly Schedule for more details.
- Heat kill the digests for 20 minutes at 80°C.
- Store digested DNA in the refrigerator (4°C)for use in the very near future.
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.
For more information you can check out the other restriction digest protocols.
or instead, discuss this protocol.
Following is the Richard Lab:Restriction Digest protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
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