Protocol: Restriction Digests (wear gloves, goggles, lab coat)
This protocol is written for 6 student groups.
Instructor- Advance preparation (day of experiment)
1. Aliquot 10 l of each DNA sample (CS, S1, S2, S3, S4, S5) into separate microtubes.
Label each tube (CS, S1, etc). Each student group should receive 1 DNA sample.
2. Aliquot 10 l restriction enzyme mix (EcoRI, PstI) into each of 6 different microtubes.
Label each tube “ENZ” for enzyme. Store these tubes on ice until they are used. Each
student group should receive 1 microtube of enzymes.
3. Aliquot 5 l gel loading dye into each of 6 different microtubes. Label each tube “DYE”.
Each student group should receive 1 microtube of loading dye.
4. Set temperature block at 37oC.
Students1. Using a P20 micropipettor and a fresh tip for each sample, pipet 10 μl of the enzyme
(ENZ tube on ice) to the bottom of each DNA sample.
2. Tightly cap the tubes and mix the components (DNA + enzymes) by gently flicking the
tubes with your finger.
3. With the help of the instructor, quickly spin the tubes in a microcentrifuge to move the
contents to the bottom of the tubes.
4. Place the tubes in the temperature block or waterbath set at 37oC and allow to the
digestion to occur for 45 minutes.
5. After 45 minutes, remove the tubes from the temperature block and ask your instructor
to quickly spin the tubes in a microcentrifuge to move the contents to the bottom of the
tubes.
6. Using a P20 micropipettor, add 5 μl of the DNA loading dye (tube marked DYE) to the
bottom of each digested DNA sample.
7. The samples are now ready to load into an agarose gel (see Agarose gel
electrophoresis protocol).
Heidi Sleister