Richard Lab:Restriction Digest

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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 but check this chart to be sure.
 * 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:
 * 5μl of Buffer (usually NEBuffer 2);
 * 1μl of BSA;
 * 0.5 picomoles DNA (see DNA Quantification); Mike normally uses 10μL of miniprep or 5μL of purified PCR product.
 * Water to make 48μl.

3. 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.

4. 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.

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.

Contact

 * Mike

or instead, discuss this protocol.

BioCoder version
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

Text Output
Richard Lab:Restriction Digest protocol

Source Code
Richard Lab:Restriction Digest protocol - source code

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