Richard Lab:Restriction Digest

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Current revision (14:05, 29 March 2011) (view source)
 
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Back to [[Richard_Lab:protocols | Protocols]]
Back to [[Richard_Lab:protocols | Protocols]]
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<center>
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'''This protocol is for the typical digestion of DNA done for [[Richard Lab:Amplified Insert Assembly|Amplified Insert Assembly]]. 
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'''The [[Engineering BioBrick vectors from BioBrick parts/Restriction digest | consensus digestion protocol]] should be consulted if deviating from the procedure outlined here.'''</center>
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==Overview==
==Overview==
This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
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==Materials==
==Materials==
* Prepared DNA from miniprep, PCR, or Gel Extraction
* Prepared DNA from miniprep, PCR, or Gel Extraction
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* Restriction Endonucleases including DpnI
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* Restriction Endonucleases
** With corresponding 10X buffer.  NEBuffer 2 can be used for most applications but check [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? this chart] to be sure.
** With corresponding 10X buffer.  NEBuffer 2 can be used for most applications but check [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? this chart] to be sure.
* BSA  
* BSA  
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==Procedure==
==Procedure==
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# Quickly vortex all ingredients (Buffer, BSA, DNA) before beginning.
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1. Quickly vortex all ingredients (Buffer, BSA, DNA) before beginning.
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# Add the following in a micro-centrifuge tube:  
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2. Add the following in a micro-centrifuge tube:  
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##5μl of Buffer (usually NEBuffer 2);
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::*5μl of Buffer (usually NEBuffer 2);
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##1μl of BSA;
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::*1μl of BSA;
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##0.5 picomoles DNA (see [[DNA Quantification]]);[[user:Michael A. Speer | Mike]] normally uses 10μL of miniprep or 5μL of purified PCR product.
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::*0.5 picomoles DNA (see [[DNA Quantification]]);[[user:Michael A. Speer | Mike]] normally uses 10μL of miniprep or 5μL of purified PCR product.
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##Water to make 48μl.
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::*Water to make 48μl.
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# Vortex Enzymes and add 1μl of each to the tube.
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3. Vortex Enzymes and add 1μl of each to the tube.
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# Incubate reaction in a 37°C water bath for at least one hour.
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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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##You may have to add DpnI or Antarctic Phosphatase at some point.  See the[[Richard Lab:Bio-Brick Assembly Schedule|Bio-Brick Assembly Schedule]] for more details.
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4. Incubate reaction in a 37°C water bath for at least one hour.
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# Heat kill the digest for 20 minutes at 80°C.
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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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# 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.
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::*See the [[Richard Lab:Bio-Brick Assembly Schedule|Bio-Brick Assembly Schedule]] for more details.
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# Store digested DNA in the freezer (-20°C).
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5. Heat kill the digests for 20 minutes at 80°C.
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6. Store digested DNA in the refrigerator (4°C)for use in the very near future.
==Notes==
==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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*DpnI eliminates the background from your PCR template
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*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.
*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
*Longer digest gives more complete digestion, especially if you have >1µg of DNA, but can sometimes give nonspecific digestion

Current revision

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This protocol is for the typical digestion of DNA done for Amplified Insert Assembly.

The consensus digestion protocol should be consulted if deviating from the procedure outlined here.

Contents

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.

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.

References

For more information you can check out the other restriction digest protocols.

Contact

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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