Restriction Digests

Digest 10 ug maize DNA per single-layer gel lane:

You can digest DNA for several wells in one tube. Determine the total amount (ug) of each genotype to be digested with an enzyme in a single tube: total ug DNA = (amount of DNA per lane) x ( number of lanes of genotype).

Determine the total volume of reaction per tube. You can load ~45 ul in a deep 27 well gel, 35 ul in a 30 well gel, and 30 ul in a 32 well gel. Keep in mind that you also have to add loading dye to each reaction before loading into the gel. calculations below are for 30 ul reaction volume.

Calculate a bulk digestion mix containing the total volume of ddH2O, buffer, and enzyme needed for the total number of DNA samples to be digested by the same enzyme. Prepare ~10% more than needed to allow for pipetting errors.

Label tubes for the reactions, and add the proper amount of DNA to each tube.

Prepare the bulk mix on ice, adding enzyme last. Mix well (do not vortex).

Aliquot bulk mix into reaction tubes. Mix by inversion (do not vortex).

Incubate at 37 C for 3-5 h. Place tubes at 65 C for 10 min. to inactivate enzymes.

Add loading dye to each tube, mix, quick spin.