This method is not only simple and robust enough to be used by inexperienced undergraduates, but also provides extensive pipetting practice and a basic understanding of DNA isolation. The procedure takes us ~2.5 hours for a set of 24 samples, the number of tubes accommodated by our microcentrifuge. Students can work on several sets at once by starting a new set while the samples from the first set are incubating at 60° (step 3). Two sets of samples can thus be completed in ~3.5 hours. It is most efficient to base the number of samples per set on the capacity of your microcentrifuge.
2) Grind with drill-mounted plastic pestle for approximately 30 - 40 seconds. Rinse pestle with H2O between each sample.
3) Incubate in 60 C water bath for 1 hour (30 minutes — 2 hours is OK). Meanwhile, label 0.7 ml tubes for the next step.
4) Spin at full speed (14,000x g) for 15 minutes (room temperature or 4 C). Transfer 150 mL supernatant to a 0.7 ml tube, avoiding the solid debris.
5) Add 150 mL 5M ammonium acetate. Add 300 mL isopropanol and gently mix by inverting the entire rack of tubes.
6) Incubate at room temperature for 15 min. (Tubes can be left overnight at 4 C if necessary.)
7) Spin for 5 min at 9300x g at room temperature.
8) Aspirate the supernatant.
9) Wash pellet by adding 200 mL cold (-20 C) 70% ethanol, spin again (5 min at 9300x g at room temperature) and aspirate supernatant.
10) Air dry for 15 min.
11) Add 200 mL TE, pH 8.0. (Do not vortex!)
12) Incubate at 65 C for 5 min to help dissolve DNA.
13) Done! Store samples at -20 C.
14) We use 5 mL of this DNA prep in a 20 mL PCR reaction; running half
of this reaction on a gel results in consistent, strong bands.
Sampling: More than 20 - 40 mg of leaf tissue does not improve the results. We do not weigh the samples, but estimate based on sample size ("a pinch-full"). However, it is important to stay fairly consistent. Seedling leaves are easier to grind than older tissue. Therefore, when possible, we use seedlings. Tissue samples can be extracted immediately after collection or stored at -20 C for several weeks. Longer storage increases the difficulty of grinding the samples. We use an Eppendorf Repeater Plus pipetter to rapidly dispense each solution into a set of tubes. To conserve disposables, we label a set of Repeater tips for each solution; these tips are rinsed several times with double distilled H2O after finishing a set of preps, and can be reused multiple times.
Grinding: Samples are ground with a disposable Kontes pellet pestle (Fisher cat. # K7495211590) mounted in a standard 10mm drill in place of a drill bit. Each pestle lasts for ~10-15 sets of preps. The drill is mounted on a ring stand with the drill/pestle pointing down; a set of ear protectors minimizes exposure to the noise of the drill. While grinding the tissue, we press the tube against the pestle using moderate pressure. However, the tubes and pestle can get hot, so we grind in alternate segments of pressing and slight release. The tissue should be about 80% ground-up, but the grinding time depends on the age of the leaf, with older leaves taking longer. To prevent cross-contamination between samples in a set, the pestle is rinsed by a short burst of "drilling" in a beaker of water and dried with a Kimwipe between each sample. We have not noticed any cross-contamination when using this simple precaution.
Incubation and transfer: Ground samples can be incubated at 60 C from 30 minutes to 2 hours, depending on what is convenient. We have not observed any improvement in yield of DNA upon longer incubation. Following the incubation and spin, supernatant from each tube is transferred using a fresh tip. Tilting the tube while withdrawing supernatant helps keep the tip free of debris.
Precipitation, resuspension and storage: After addition of ammonium
acetate and isopropanol, the samples are gently inverted several times
in a rack covered with a piece of cardboard. If convenient, the tubes can
be stored overnight at 4 C prior to the precipitation spin. After the spin,
we carefully remove the supernatant from each tube using a benchtop aspirator
with a 200 mL pipet tip on the end. (This speeds up the process significantly,
but be careful not to aspirate the pellet!) The aspirator is also used
to remove the ethanol wash. Alternatively, the samples can be left overnight
in ethanol at -20 C. After drying, the pellets are resuspended in 200 mL
TE, pH 8.0. The 5-minute incubation at 65 C helps speed up resuspension;
samples can be carefully pipetted up and down if additional mixing is needed.
However, there is often a small amount of undissolved debris in the tube;
this does not seem to inhibit the subsequent PCR reaction. Samples can
be frozen at -20 C for at least several years.
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