Extraction of DNA from Watermelon Leaves

by Gabriele Gusmini and Todd C. Wehner
Department of Horticultural Science
North Carolina State University
Raleigh, NC 27695-7609

Levi and Thomas (1999)developed an improved procedure for the extraction of high-quality DNA from a large sample of watermelon leaves. This technique is suitable for isolating high quality DNA used in RFLP and AFLP marker analysis employing restriction enzymes. However, RAPD and SSR marker analysis do not require high quality genomic DNA, so a modified DNA extraction procedure suitable for isolating genomic DNA from a large number of small leaf samples might be sufficient for SSR marker analysis. On this web-page, we report an alternative DNA extraction procedure from a large number (24 samples per run) of small watermelon leaf samples (≈500 mg) in 1.5 ml micro-centrifuge tubes.

Levi, A., and C. Thomas. 1999. An improved procedure for isolation of high quality DNA from watermelon and melon leaves. Cucurbit Genetics Cooperative Report 22:41-42.

Leaf collection and storage

Young (1-4 day old) and tender leaves (quantity 4-6) should be collected. Three procedures for storing the leaf samples in -80°C, before DNA extraction, are possible:

Each sample collected in a polyethylene Easy Zipper Ziploc® Bag resistant to freezing temperatures and stored in a -80°C freezer.
Entire leaves harvested, as described by Levi and Thomas (2), ground with three rounds of liquid nitrogen to a fine powder, and stored in 50 ml polyethylene tubes in a -80°C freezer.
Entire leaves harvested, 2-4 leaf blade squares (about 5 X 5 mm) cut with scissors or razor blade from the leaves, and stored in a 1.5 ml microcentrifuge tube kept in a -80°C freezer.

We consider the second and third procedures to be the most suitable for long-term storage and sub-sampling of a large number of leaf samples used with the DNA extraction procedure presented herein. These sampling procedures allow DNA extraction from small tissue samples, while avoiding repeated thawing and oxidation of leaf tissue during sub-sampling.

Extraction buffer

In a 50 ml polypropylene tube add:
- 25 ml of extraction solution
  - 0.1 M Tris-base
  - 0.5% Sarcosyl
  - 1.4 M NaCl
  - 20.0 mM EDTA-Disodium
  - 2.5% CTAB
- 250 mg of Polyvinylpyrrolidone, molecular weight 40,000 (Soluble PVP or PVP-40)
- 250 mg Polyvinylpolypyrrolidone (Insoluble PVP or PVPP)
Incubate for 10-15 minutes at 60°C and mix vigorously
After incubation add 125 microl of Mercaptoethanol
Incubate at 60°C

DNA extraction procedure

Add 700 microl of extraction buffer to 50-100 mg of leaf tissue (intact or previously ground with liquid nitrogen) in 1.5 ml microcentrifuge tubes; use P1000 pipette tips, cut at 1/3 of their length.
Homogenize each sample:
- If starting from intact leaf tissue, add quartz or glass sand (the tip of a spatula) and homogenize using a Kontes™ Pellet Pestle™. In this case, it is very important to use tissue from very young leaves (1-4 days).
- If starting from leaf tissue previously ground with liquid nitrogen, vortex gently.
Incubate for 30 minutes at 60°C and vortex vigorously every 10 minutes.
Add to each sample 500 microl of Chloroform:Isoamyl Alcohol (24:1).
Vortex vigorously until the mix color turns homogeneous and light-green; release gas and reseal the cap.
Centrifuge for 5 minutes at 12,500 rpm.
Transfer the supernatant from each sample (≈500 microl) to new 1.5 ml tubes.
Add to each sample 500 microl (or 1 volume) of ice-cold Isopropanol.
Mix gently by inversion.
Incubate for 20 minutes at -20°C.
Centrifuge for 15 minutes at 12,500 rpm.
Pour supernatant and suspend the pellets in 500 microl 70% ethanol.
Centrifuge for 15 minutes at 12,500 rpm.
Pour supernatant and dry the pellets at room-temperature then suspend in 100 microl of 0.1X TE.
Centrifuge for 5 minutes at 12,500 rpm.
Transfer the supernatant from each sample to a new container for final storage (0.5 ml microcentrifuge tubes or 96-well plates).