Richard Lab:Restriction Digest
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This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
This is what Mike calls "Standard Assembly 2.0" and is a method of "bio-bricking" two parts together. For more information on bio-bricking see this link. This method combines the ease of standard assembly with the fidelity of triple-antibiotic (3A) assembly. The basic steps are as follows. Your two parts will be thusly labeled "insert" and "vector" although initially they will both be contained on separate plasmids.
- Miniprep both "insert" and "vector" from their respective cultures.
- PCR the insert plasmid using a high-fidelity polymerase and the same primers you use for colony PCR.
- Purify the PCR product.
- Digest the both vector and PCR'd insert according to the instructions in this protocol.
- Add DpnI restriction endonuclease to the "insert" digest.
- Add Antarctic Phosphatase to the "vector" digest.
- 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.
- BSA (optional)
- 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
- 0.5 picomoles DNA (see DNA Quantification)
- Water to make 48μl
- Vortex Enzymes and add 1μl (20 units) of each to the tube.
- Incubate reaction in a 37°C water bath for at least one hour.
- Heat kill the digest for 20 minutes at 80°C.
- If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes at then heat kill againat 75°C for 15 min.
- Store digested DNA in the freezer (-20°C).
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- 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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