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 protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
<center>'''-----EcoRI--XbaI--Part--SpeI--PstI-----'''</center>
<center>'''-----EcoRI--XbaI--Part--SpeI--PstI-----'''</center>
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==Materials==
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* Prepared DNA from miniprep, PCR, or Gel Extraction
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* Restriction Endonucleases
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** With corresponding 10X buffer.  NEB buffer 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.
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* BSA (optional)
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* Phosphatase
==Procedure==
==Procedure==
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==References==
==References==
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If you are needing to know more then you can check out the [[Restriction_Digest | other restriction digest protocols]].
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For more information you can check out the [[Restriction_Digest | other restriction digest protocols]].
==Contact==
==Contact==

Revision as of 15:32, 12 June 2009

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-----

Materials

  • Prepared DNA from miniprep, PCR, or Gel Extraction
  • Restriction Endonucleases
    • With corresponding 10X buffer. NEB buffer 2 can be used for most applications but check this chart to be sure.
  • BSA (optional)
  • Phosphatase

Procedure

  1. Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning
  2. Add the following in a micro-centrifuge tube
    1. 5μl of Buffer 2
    2. 1μl of BSA
    3. 42μl of DNA solution (Dilute PCR products in half)
  3. Vortex Enzymes and add 20 units (1μl) of each to the tube
  4. Incubate reaction in a 37°C water bath for at least one hour (I like 1:45).
  5. Heat kill the digest for 15 minutes at 75°C.
  6. 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.
  7. Store digested DNA in the freezer (-20°C).

Notes

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

References

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

Contact

or instead, discuss this protocol.

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