Restriction digest

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==Specific Protocols==
==Specific Protocols==
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[[Enzyme Selection for BioBrick Digest]]  
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[[Enzyme selection for BioBricks digest]]  
[[Endy:Restriction Digest]]
[[Endy:Restriction Digest]]
[[Knight:Restriction Digest]]
[[Knight:Restriction Digest]]
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[[Silver: Restriction Digest]]
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[[Griffitts:Restriction digest]]
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[[Richard Lab:Restriction Digest]]
==Notes==
==Notes==
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*For help with specific enzymes, see [[Restriction enzymes]]
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*For buffer composition, see [[Restriction digest/Buffers]]
*If you are interested in cutting near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA.  So include extra bases to increase the efficiency of cutting.  Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully).  Thus, to be on the safe side, use 8 bases whenever possible. [http://www.neb.com/ NEB] has more information [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp here].  Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.
*If you are interested in cutting near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA.  So include extra bases to increase the efficiency of cutting.  Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully).  Thus, to be on the safe side, use 8 bases whenever possible. [http://www.neb.com/ NEB] has more information [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp here].  Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.
*Tom was once having some trouble with failed restriction digests.  He called NEB and they recommended doing the digest in a larger volume.  Therefore, the Knight lab typically does digests in either 50 μL or 100 μL volumes rather than the 20 μL volume that the Endy lab uses.  If the sample needs to be concentrated, some method of [[Purification of DNA | DNA purification]] is used.
*Tom was once having some trouble with failed restriction digests.  He called NEB and they recommended doing the digest in a larger volume.  Therefore, the Knight lab typically does digests in either 50 μL or 100 μL volumes rather than the 20 μL volume that the Endy lab uses.  If the sample needs to be concentrated, some method of [[Purification of DNA | DNA purification]] is used.
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*Improving the efficiency of [[EcoRI/SpeI Double Digest]].
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*'''Reaction Volumes''' - I used to have very inconsistent results when digesting with EcoRI, using the 20μl reaction volume. Using the Knight Lab's 50μl mix has recently given good results.  I haven't tested this enough to say it is a statistically significant result.  --[[User:Bcanton|BC]] 13:39, 2 Jun 2005 (EDT)
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[[Category:Protocol]]  
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[[Category:In vitro]]  
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[[Category:DNA]]

Current revision

Contents

General Information

Restriction digest involves the cutting of DNA at specific recognition sequences by enzymes.

General Procedure

Specific Protocols

Enzyme selection for BioBricks digest

Endy:Restriction Digest

Knight:Restriction Digest

Silver: Restriction Digest

Griffitts:Restriction digest

Richard Lab:Restriction Digest

Notes

  • For help with specific enzymes, see Restriction enzymes
  • For buffer composition, see Restriction digest/Buffers
  • If you are interested in cutting near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA. So include extra bases to increase the efficiency of cutting. Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully). Thus, to be on the safe side, use 8 bases whenever possible. NEB has more information here. Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.
  • Tom was once having some trouble with failed restriction digests. He called NEB and they recommended doing the digest in a larger volume. Therefore, the Knight lab typically does digests in either 50 μL or 100 μL volumes rather than the 20 μL volume that the Endy lab uses. If the sample needs to be concentrated, some method of DNA purification is used.
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