|
Digest 10 ug maize DNA per single-layer
gel lane:
1. 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).
2. 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.
Stock |
Final Conc. |
10 ug DNA |
ddH2O |
-- |
14.5 ul |
10X Buffer |
1X |
3 ul |
10 U/ul Enzyme |
2.5 U/ug DNA |
2.5 ul |
1 ug/ul DNA |
-- |
10 ul |
3. 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.
4. Label tubes for the reactions, and add the proper amount of DNA
to each tube.
5. Prepare the bulk mix on ice, adding enzyme last. Mix well (do
not vortex).
6. Aliquot bulk mix into reaction tubes. Mix by inversion (do not
vortex).
7. Incubate at 37 C for 3-5 h. Place tubes at 65 C for 10 min. to
inactivate enzymes.
| Note: if the concentration of DNA is low,
you may have to precipitate and re-suspend the DNA in the
right amount of TE after digestion. Perform digestion as indicated
above. Stop reaction by adding 5 M NaCl to a final concentration
of 0.25 M NaCl. Add 2.5 volumes of EtOH, mix well, place at
-80 C for 30 min. or at -20 C overnight. Precipitated DNA
can be stored in EtOH at -20 C for an indefinite amount of
time. Centrifuge in microfuge at full speed for 10 min. Pour
off supernatant, drain tubes and allow EtOH to completely
evaporate. Dissolve pellet in the proper amount of TE for
loading into a well of agarose gel. |
8. Add loading dye to each tube, mix, quick spin. |
