Genomic DNA Miniprep Protocol - Plant

Before Start:
  • Water bath equilibrated to 65 oC.
  • Equilibrate sterile ddH2O or Elution Buffer at 65 oC.
  • Use extreme caution when handling liquid nitrogen.

Fresh/frozen specimens: Allow more efficient recovery of DNA. However, due to the tremendous variation in water and polysaccharide content of plants, sample size should be limited to200 mg. Best results are obtained with young leaves or needles. To prepare samples, collect tissue in a 1.5 mL or 2 mL microcentrifuge tube and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube. Grind the tissue using disposable Kontes pellet pestles, which are available from VWR (Cat#KT749521-0500). Alternatively, one can allow liquid nitrogen to evaporate andthen store samples at -70oC for later use.

Dried specimens: This is the most robust method for isolation of total cellular (mitochondrial,chloroplast, and genomic) DNA. Yields are usually sufficient for several tracks on a Southern blot for RFLP mapping. Drying allows storage of field specimens for prolonged periods of time prior to processing. Samples can be dried overnight in a 45 oven, powdered, and stored dry at room temperature. To prepare dried samples, place ≦50 mg of dried tissue into a 2.0 mL microcentrifuge tube and grind using a pellet pestle.

For critical work such as PCR and cloning, pestles are best used a single time then soaked in a dilute bleach solution immediately after use until cleaning. Disposable pestles may be autoclaved several times. For standard Southern analysis, the same pestle can be reused several times to grind multiple tissue samples by rinsing with ethanol and carefully wiping the surfaces clean between samples.

  1. To 10-50 mg (dry) or 100 mg (wet) powdered tissue, add 600 μL Buffer PL1 in a 2.0 mL centrifuge tube. Optional: Add 10 μL β-mercaptoethanol and vortex vigorously to mix. Make sure to disperse all clumps. Tip: Process in sets of four to six tubes: grind, add Buffer PL1 and β-mercaptoethanol, and proceed to Step 2 before starting another set. Initially, do not exceed 50 mg dried tissue. Amount can be increased according to results.

  2. Incubate at 65 oC for 10 min. Mix sample twice during incubation by inverting tube. Optional: If necessary, add 5 μL RNase A into the lysate before incubation to remove the RNA.

  3. Add 140 μL Buffer PL2 and mix well by vortexing for 10s. Centrifuge at 13,000 rpm for 10 min.

  4. Carefully transfer the supernatant to a clean 2.0 mL tube. Add 0.5 volume of Buffer PL3 and 1 volume 100% ethanol and mix well by vortexing for 10s.

  5. Apply the entire sample (including any precipitate that may have formed) to a DNA column placed in a 2.0 mL collection tube (supplied). Centrifuge the column at 13,000 rpm for 1 min. Discard the flow- through liquid with the collection tube.

  6. Transfer column to a new collection tube and add 600 μL DNA Wash Buffer. Centrifuge at 13,000 rpm for 1 min and discard the flow-through liquid. Reuse the collection tube. Note: Wash Buffer must be diluted with absolute (96%-100%) ethanol prior to use. Follow directions on label.

  7. Repeat Step 6. Discard flow-through and collection tube, use another 2.0 mL collection tube in next step.

  8. Place the column, with the lid open, into a new collection tube and centrifuge at 13,000 rpm for 2 min. This step is critical for removing residual ethanol that may otherwise be eluted with DNA and interfere with downstream applications.

  9. Transfer column to a clean 1.5 mL tube. Add 100 μL Elution Buffer (or sterile deionized water) pre-warmed to 65 oC and incubate at room temperature for 3 to 5 min, centrifuge at 13,000 rpm for 1 min to elute DNA. Smaller volumes will significantly increase DNA concentration but give lower yields. Use of more than 200 μL of buffer for elution is not recommended.

  10. Repeat Step 9 with another 100 μL Elution Buffer. This may be performed using another 1.5 mL tube to maintain a higher DNA concentration in the first eluate. Tip: To increase DNA concentration, add buffer and incubate the column at 60-70 oC- for 5 min before elution. Total DNA yields vary depending on type and quantity of sample. Typically, 10-50 μg DNA with an A260/A280 ratio of 1.7-1.9 can be isolated using 50 mg dried tissue.

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