DNA restriction digest

From 2008.igem.org

Contents

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)
  • Matching NEB Buffer for the Restriction Enzyme
NEBuffer 1 NEBuffer 2 NEBuffer 3 NEBuffer 4
EcoRI 100% 100% 100% 100%
XbaI 0% 100% 75% 75%
SpeI 75% 100% 25% 75%
PstI 75% 75% 100% 50%
  • 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)

<under construction>

Prefix Insert (EcoRI/SpeI)

<under construction>

Notes

  • Remember to check that REs are compatible.
  • Reaction can also be stopped by adding 5μl 10X loading buffer.
  • <10% of digest should be RE
  • REs are stored in glycerol. <5% glycerol should be present in digest. Therefore, a minimum 10-fold dilution is necessary so the enzymes don't act funky.
  • It's always a good idea to gently shake and spin down RE and RE buffer prior to use.
References: Short Protocols in Molecular Biology. Vol. 1. 5th edition. 3-3 to 3-4.
NEB

References

  1. http://openwetware.org/wiki/Restriction_digest
  2. http://openwetware.org/wiki/Endy:Restriction_Digest
  3. http://openwetware.org/wiki/Knight:Restriction_Digest
  4. http://2008.igem.org/Team:Hawaii/Initial_Synth._Oligo_Assembly
  5. Short Protocols in Molecular Biology. Vol. 1. 5th edition. 3-3 to 3-4.
  6. New England BioLabs Restriction Enzyme Product Insert

Contact

  • Grace Kwan

or instead, discuss this protocol.