Watermelon
Crop Information
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).
