Gawel-Jarret1991 Article AModifiedCTABDNAExtractionProc

Plant Molecular BiologyReporter pages 262-266
Volume 9(3) 1991
Protocol
A Modified CTAB D N A Extraction Procedure

for Musa and Ipomoea
N.J. Gawel and R.L. Jarret
(NJG) Department of Plant Introduction, and (RLJ) USDA-ARS Regional Plant

Introduction Station, Georgia Station, 1109 Experiment St., Griffin, GA 30223,
USA
Key Words: DNA extraction, CTAB, Ipomoea,Musa, sweetpotato, banana, okra,

pigeon pea, cotton.
Abstract: The utilization of current nucleic acid technologies in crop improve-

ment and phylogenetic studies require the development and application of
efficient DNA extraction procedures from plants. This paper describes efficient,
reliable DNA extraction procedures for lpomoeaand Musa. These procedures are
combinations and modifications of the techniques described by Murray and
Thompson (1980) and Saghai-Maroof et al. (1984) and are applicable, without
further modification, to other plant genera.
he applications of current nucleic acid technologies in crop im-
T provement are numerous: gene introgression, gene mapping,

genetic fingerprinting, phylogenetic analyses, etc. (Burr et al.,
1986). These techniques have numerous applications to improvement of
Musa (Jarret, 1990; Gawel and Jarret, 1990) and Ipomoea research (Jarret
and Florkowski, 1990). The development of efficient DNA extraction
techniques, which yield DNA of a purity adequate for restriction enzyme
digestion, has proven difficult from plant materials in these genera. After
experimenting with a variety of techniques, our lab has developed
Abbreviations: RT, room temperature; CTAB, hexadecyltrimethylammonium

bromide.
CTAB DNA Extraction 263
simple, efficient procedures for the extraction of Musa and Ipomoea DNA.
We have, on occasion, received inquiries from other laboratories con-
cerning these techniques. Therefore, with the hope of helping others to
avoid the frustrations we initially encountered, we present our protocol
for isolation of high molecular weight DNA from Musa and lpomoea leaf
tissue. These procedures are a combination of modified extraction
techniques of Murray and Thompson (1980) and Saghai-Maroof et al.
(1984).
Materials and Methods

Reagents
Extraction buffer (EB): [1% (Ipomoea) or 2% (Musa) ( w / v ) CTAB, 100
mM Tris.HC1 pH 8.0, 1.4 M NaC1, 20 mM EDTA, 0.1% (Musa) or
1.0% (Ipomoea) (v/v) mercaptoethanol (ME a d d e d immediately
prior to use)]
TE: 10 mM Tris, 1.0 mM EDTA, pH 8.0
Precipitation buffer (PB): EB without ME or NaC1
EtOH: 100% and 70%
Isopropanol
Chloroform: Isoamyl alcohol (24:1, v / v )
RNAse
D N A Extraction from Musa

9 Grind lyophilized leaf tissue (500 mg) in liquid N2wit h a mortar and
pestle or with a grinding mill.
9 Add the powdered tissue to 20 mL pre-heated extraction buffer and
incubate (65~ for 30 rain with occasional mixing.
9 Add 15 mL of chloroform:isoamyl alcohol (24:1, v/v), and mix by
inversion for 15 rain.
9 Centrifuge 5 min at 5,000 x g at room temperature (RT) and filter the
aqueous phase through Miracloth (or equivalent).
9 Add an equal volume of ice-cold isopropanol. Mix by inversion
until DNA precipitates (10 to 20 seconds).
9 Hook the DNA onto a bent Pasteur pipet or spool onto a glass rod
and rinse in a beaker of 70% ethanol.
9 Blot the D N A dry on a Kimwipe or paper towel.
9 Dissolve the D N A i n 250 to 500 iLL TE buffer ( Extracts may need to
be heated at 65~ for a short time to completely dissolve.)
9 Add RNAse (final concentration 10 ~g/mL) and incubate30 min at
room temperature.
9 Add 1/10 vol 3 M NaOAc (pH 6.8) and 2 volumes of 95% ethanol.
264 Gawel and Jarret
9 H o o k D N A on a bent Pasteur pipet, rinse in 70% alcohol and blot

dry.
9 Dissolve in 250 to 500 I.tL TE.
D N A Extraction from Ipomoea

9 A d d 200 m g p o w d e r e d lyophilized leaf tissue to 8 m L EB con taining
1% ( w / v ) CTAB and 1% ( v / v ) ME (see above).
9 Incubate the mixture for 30 rain at 65~
9 Extract with an equal v o l u m e of chloroform:isoamyl alcohol.
9 Centrifuge at 2,500 x g for5 rain at r o o m t e m p e r a t u r e to separate the
phases.
9 Draw off the a q u e o u s phase and re-extract with an equal v o l u m e of
chloroform:isoamyl alcohol.
9 Centrifuge at 5,000 x g and transfer the u p p e r phase to a 15 m L Corex
tube.
9 A d d an equal v o l u m e of PB and mix b y quick inversion several
times.
9 Allow to sit at r o o m t e m p e r a t u r e for I hour.
9 Pellet the nucleic acids b y centrifugation at 5,000 x g at RT.
9 Redissolve the pellet in 450 ~L 1M NaC1 with heat (1 hr at 65~
9 The D N A is precipitated b y addition of 2 v o l u m e s of 100% EtOH,
c o l l e c t e d b y c e n t r i f u g a t i o n , w a s h e d w i t h 70% E t O H a n d
r e s u s p e n d e d in 200 t.tL TE.
Discussion
Musa
With this technique, we generally obtain 100 to500 ~tg D N A (A2~0/A~0 =
1.85-1.95) per g r a m fresh weight leaf tissue (Table I). Best results are
obtained w h e n the leaf tissue is harvested as y o u n g as possible, prefer-
ably while still tightly furled. The midrib should be r e m o v e d prior to
lyophilization. If lyophilization is not possible, fresh tissue m a y be used
if the CTAB concentration is increased to 4% ( w / v ) . W h e n using fresh
tissue, care should be taken to assure that the g r o u n d tissue does not thaw
before it is a d d e d to the extraction buffer. The use of 0.1% mercaptoethanol
does not c o m p l e t e l y inhibit oxidation in all cases. H o w e v e r , the oxida-
tion which does occur d o e s not a p p e a r to affect the activity of restriction
enzymes. If m o r e than 0.1% m e r c a p t o e t h a n o l is used, D N A r e c o v e r y is
greatly reduced. In addition, care should be taken to h o o k out the D N A
i m m e d i a t e l y after the isopropanol precipitation in o r d e r to avoid the co-
precipitation of contaminating proteins.
CTAB D N A Extraction 265
Table I. Yields and A260/A~0 values for D N A extracted from various plant
genera.
Plant Lyophilized tissue D N A Yield A260/A2s o
Musa acuminata 500 mg 393.4 ~g* 1.87"

lpomoea batatas 200 mg 249.2 gg* 2.08*
Gossypium hirsutum 500 mg 276.6 ~tg* 2.01"
Juglans nigra 1000 mg 380.0 ~tg* 1.97"
Abelmoschus manihot 250 mg 198.8 ~tg** 2.10"*
Cajanus cajan 250 mg 445.0 ~g** 2.02**
*Mean of 10 extractions.
**Single extraction.
Ipomoea Musa Abel

E HBMKEHBM K EHB M
Fig. 1. Restriction e n z y m e digestion of D N A extracted from Ipomoea batatas,

Musa acuminata, and Abelmoschus manihot. Restriction enzymes used are Eco
RI (E), HindllI (/-/), Barn HI (B)and MspI (M). Molecular weightmarkers are Hind
Ill-digested X. The M. acuminata DNA has been treated with RNAase.
266 Gawel and Jarret
Ipomoea
We routinely separate the DNA resuspended in TE from insoluble
material by centrifugation in a microcentrifuge for 30 to 60 sec. This
improves the efficiency of the e n z y m e digestion. Yields range from 100
to 500 ~g D N A / 2 0 0 m g lyophilized leaf tissue following this procedure.
However, yields were noticeably higher w h e n using sweetpota to (Ipcrnoea
batatas) as compared to other lpomoea species.
Yields of DNA m a y vary with the physiological age of the plant
material, the period of time that the material is stored prior to extraction,
etc.; hence, modifications m a y be necessary to increase yields in specific
instances. Treatment with RNAase is not essential for complete diges-
tion by restriction enzymes. The extraction procedures described here
are relatively simple and efficient. These procedures yield high-quality
DNA which is readily digested by restriction endonucleases. We have
adapted the Murray a n d T h o m p s o n (1980) CTAB technique to other
plant genera ( Cajanus, Gossypium, Juglans, Abelmoschus ) a n d r e c o m m e n d
this technique as a starting point w h e n developing a DNA extraction
procedure for plants.
References
Burr B., S.V. Evolaand F.A. Burr. 1986. The applications of restriction fragment polymor-
phisms to plant breeding. In: Genetic Engineering: Principles and Methods. New York,
Plenum Press.
Gawel, N.J. and R.L.Jarret. 1990. Cytoplasmic genetic diversity in bananas and plantains.
Euphytica 52:19-23.
Jarret, R.L. 1990. Molecular methods for detection genetic diversity in Musa. In: Identifica-
tion of Genetic Diversity in the Genus Musa. R.L.Jarret, ed. INIBAP,Montpellier.
Jarret• R.L. and W.J. F••rk•wski.199•.•n •itr• vs•fie•d gene bank maintenance •f sweet••ta t•
germ plasm: Major costs and considerations. Hot tScience25(2):141-146.
Murray, M.G. and W.F. Thompson, 1980. Rapid isolation of high molecular weight plant
DNA. Nuc. Acids Res. 8:4321-4325.
Saghai-Maroof, M.A., K.M. Soliman, R.A. Jorgensen and R.W. Allard. 1984. Ribosomal
DNA spacer-length polyrnorphisms in barley: Mendelian inheritance, chromosomal
location, and population dynamics. Proc. Natl. Acad.Sci. (USA)81:8014-8018.

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Plant Molecular BiologyReporter pages 262-266

Volume 9(3) 1991

A Modified CTAB D N A Extraction Procedure

N.J. Gawel and R.L. Jarret

(NJG) Department of Plant Introduction, and (RLJ) USDA-ARS Regional Plant

Key Words: DNA extraction, CTAB, Ipomoea,Musa, sweetpotato, banana, okra,

Abstract: The utilization of current nucleic acid technologies in crop improve-

he applications of current nucleic acid technologies in crop im-

T provement are numerous: gene introgression, gene mapping,

Abbreviations: RT, room temperature; CTAB, hexadecyltrimethylammonium

Materials and Methods

D N A Extraction from Musa

9 H o o k D N A on a bent Pasteur pipet, rinse in 70% alcohol and blot

D N A Extraction from Ipomoea

Plant Lyophilized tissue D N A Yield A260/A2s o

Musa acuminata 500 mg 393.4 ~g* 1.87"

Ipomoea Musa Abel

Fig. 1. Restriction e n z y m e digestion of D N A extracted from Ipomoea batatas,

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