Difference between revisions of "Restriction digest"

From OpenWetWare
(Specific Protocols)
m (Specific Protocols)
(One intermediate revision by the same user not shown)
Line 16: Line 16:
[[Griffitts:Restriction digest]]
[[Griffitts:Restriction digest]]
[[Richard Lab:Restriction Digest]]

Revision as of 13:41, 12 June 2009

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


  • 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.