DNA restriction digest

Overview
Restriction digest of DNA for downstream electrophoresis (to verify fragment existence) or BioBrick ligation assembly

Materials
List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.


 * Restriction Enzyme (from NEB)
 * EcoRI
 * XbaI
 * SpeI
 * PstI
 * Matching NEB Buffer for the Restriction Enzyme
 * X μL DNA (usually ~500 ng depending on downstream uses).
 * 1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.)
 * 1 μL BioBricks enzyme 2
 * 15.6 μL deionized, sterile H2O
 * 0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)

Procedure
Postfix Vector (SpeI/PstI)
 * 1) Combined 0.4 μl DNA (from plasmid prep), 15.6 μl nanopure water, 2 μl 10X NEBuffer 2, 1 μl SpeI, and 1 μl PstI.
 * 2) Incubated 2.5 hours at 37C (recommended incubation of at least 1 hour)
 * 3) Stopped reaction by incubation 10 min. at 65C.

Postfix Insert (XbaI/PstI)
 * 1) Combined 0.4 μl DNA (from plasmid prep), 15.6 μl nanopure water, 2 μl 10X NEBuffer 2, 1 μl XbaI, and 1 μl PstI.
 * 2) Incubated 2.5 hours at 37C (recommended incubation of at least 1 hour)
 * 3) Stopped reaction by incubation 10 min. at 65C.

Prefix Vector (EcoRI/XbaI)

Prefix Insert (EcoRI/SpeI)

Contact

 * Grace Kwan

or instead, discuss this protocol.