BioTechniques

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Modified CTAB Protocol Using a Silica Matrix for

Isolation of Plant Genomic DNA

Junchao Huang, Xuejun Ge & Mei Sun

To cite this article: Junchao Huang, Xuejun Ge & Mei Sun (2000) Modified CTAB Protocol
Using a Silica Matrix for Isolation of Plant Genomic DNA, BioTechniques, 28:3, 432-434, DOI:
10.2144/00283bm08

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Published online: 29 Aug 2018.

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Benchmarks
protocol, we found that we could omit Biochim. Biophys. Acta 107:144-145. from many woody plant species that
the use of Fairbanks B and Fairbanks C 4.Sambrook, J., E.F. Fritsch and T. Maniatis. contain high levels of polysaccharides
Molecular Cloning: A Laboratory Manual.
solutions. This elimination speeds up (2nd ed.) CSH Laboratory Press, Cold Spring
and/or secondary metabolites. Such
the processing time to about 15 min Harbor, NY. polysaccharides have solubility proper-
with only a twofold sacrifice of sensi- 5.Sreeramulu, G. and N.K. Singh. 1995. ties similar to those of DNA and inter-
tivity. It also reduces the number of so- Destaining of Coomassie brilliant blue R-250- fere with the isolation of pure DNA
lutions that need to be prepared from stained polyacrylamide gels with sodium when ethanol or isopropanol is used to
chloride solutions. Electrophoresis 16:362-
four to only two. Bringing the solution 365.
precipitate the DNA.
containing the gel to the boiling point 6.Syrovy, I. and Z. Hodny. 1991. Staining and Complex or time-consuming proce-
appears to be a prerequisite for the im- quantification of proteins separated by poly- dures such as the cesium chloride den-
proved sensitivity and speed of our acrylamide gel electrophoresis. J. Chromatog. sity gradient technique are frequently
569:175-196.
method. Our method is quite forgiving, 7.Wu, W. and M.J. Welsh. 1995. Rapid
used to surmount this problem. But this
however, and all steps can be per- Coomassie blue staining and destaining of procedure is not suitable for genetic
formed for longer periods. As long as polyacrylamide gels. BioTechniques 20:386- analyses in population studies, where
the gels are not boiled to near dryness, 388. large numbers of samples are often
the actual boiling times are not impor- 8.Merril, C.R. 1990. Guide to Protein Purifica- used. Glass or silica can specifically
tion. Methods Enzymol. 182:477-488.
tant. Evaporation of the isopropanol bind nucleic acids in the presence of a
presumably occurs early in the boiling We are grateful to Dr. Hans Wiech for
chaotropic salt (5). These materials
process, so for consistent results, we pointing out the original Fairbanks refer-
may be alternatives to ethanol or iso-
avoid reusing the solutions. Though ence. This work was supported by NIH,
propanol for DNA precipitation. They
longer boiling or rinsing times do not PEW and BMBF grants to J.C.A.B. Address
have been widely used for DNA frag-
reduce sensitivity, they also do not im- correspondence to Ursula Jakob, Depart-
ment binding, as in cleaning up PCR
prove it. The times listed have been op- ment of Biology, University of Michigan,
products. The purity of the recovered
timized for speed. Ann Arbor, MI 48109-1048, USA. Internet:
DNA is high and adequate for most
Silver staining is another commonly [email protected]
molecular analyses.
used technique with extremely high In this study, we describe a simple,
sensitivity. However, unlike our pro- Received 12 July 1999; accepted 6 De- efficient minipreparation procedure for
cess, it involves the use of toxic chemi- cember 1999. genomic DNA extraction from recalci-
cals, its process is lengthy, complicated trant plant species, such as some man-
and unforgiving. Moreover, it stains Connie Wong, Srinavas Srid- grove species and sweet potato. The
different proteins to different degrees, procedure combines the advantages of
depending on the protein’s silver-bind- hara, James C.A. Bardwell the high DNA yield using the CTAB
ing property, making silver less suited and Ursula Jakob method with the high DNA purity from
for protein quantification. Fluorescent University of Michigan cleaning up silica. The plant species
reagents such as fluorescamine and o- Ann Arbor, MI, USA Amaranthus tricolor L. was used to op-
phthalaldehyde are also sensitive. How- timize conditions for high quantitative
ever, their fluorescent intensity dimin- yield of DNA. The recalcitrant man-
ishes with time, and UV radiation must grove plant Bruguiera gymnorrhiza (L.)
be used to observe the staining pattern. Lamk. (Rhizophoraceae) and several
Coomassie blue remains the most pop- other plant species, including sweet
ular protein stain. In conclusion, our potato Ipomoea batatas (L.) Lam. (Con-
rapid Fairbanks method combines all volvulaceae), Sonneratia caseolaris (L.)
the features of a useful protein staining Modified CTAB Protocol Engl. (Sonneratiaceae), Sonneratia alba
technique: it is fast, highly sensitive Using a Silica Matrix J. Smith and Alpinia formosana K.
and inexpensive. Schum. (Zingiberaceae) were used to
for Isolation of Plant test the quality of extracted DNA.
Genomic DNA Reagents used in the procedure include
REFERENCES the following: homogenization buffer
(HB) containing 2% (wt/vol) CTAB,
BioTechniques 28:432-434 (March 2000)
1.Fairbanks, G., T.L. Steck and D.F.H. Wal- 100 mM Tris-HCl, pH 8.0, 1.4 M NaCl
lach. 1971. Electrophoretic analysis of the and 50 mM EDTA; extraction buffer
major polypeptides of the human erythrocyte
membrane. Biochemistry 10:2606-2617.
Of the DNA extraction methods de- (EB): chloroform: isoamyl alcohol
2.Kurien, B.J. and R.H. Scofield. 1998. Heat scribed in the literature, the cetyltri- (24:1, vol/vol); Sephaglas BP (Amer-
mediated quick Coomassie blue protein stain- methylammonium bromide (CTAB) sham Pharmacia Biotech, Piscataway,
ing and destaining of SDS-PAGE gels. Indian method (1) is widely used because it is NJ, USA); 4 M guanidine thiocyanate
J. Biochem. Biophys. 35:385-389. fast, efficient and can yield high-quality (GuSCN); and TE buffer, pH 8.5.
3.Meyer, T. S. and B.L. Lambert. 1965. Use of
coomassie brilliant blue R250 for the elec- DNA from a variety of starting materi- PCR amplifications of inter-simple
trophoresis of microgram quantities of parotid als. However, with this method, it is sequence repeat (ISSR), using UBC
saliva proteins on acrylamide-gel strips. difficult to obtain high-quality DNA primer no. 807 (5′-AGAGAGAGAGA-

432 BioTechniques Vol. 28, No. 3 (2000)

Protocol 1. DNA Extraction Procedure

1. Harvest about 0.1 g fresh leaves, transfer into a 1.5 mL Microcen-

trifuge tube (Treff AG, Degersheim, Switzerland). Place the tube on
ice.
2. Aliquot 200 µL of HB (including 0.2% mercaptoethanol) into each
tube, mix with a bit of sand. As an alternative, liquid nitrogen can be
used for hard tissue.
3. Homogenize leaf tissue with a tissue homogenizer.
4. Add 500 µL of HB to each tube, mix and incubate at 65°C for 20 min
with occasional mixing.
5. Add 700 µL of EB to each tube and mix by inversion for 1 min.
6. Centrifuge at 10 000 rpm (Model No. 5417C, Eppendorf Centrifuge,
Hamburg, Germany) for 5 min.
7. Transfer the top (aqueous) layer into a fresh tube.
8. Add 1.0 volume of 4 M GuSCN and adjust to pH 6.5 with HCl.
9. Add 15 µL silica suspension and incubate the mixture at room tem-
perature for 5 min with occasional mixing.
10. Centrifuge at 8000 rpm for 30 s and discard the supernatant.
11. Resuspend the matrix containing DNA with 500 µL of 70% ethanol.
Centrifuge at 8000 rpm for 30 s and discard supernatant. Repeat the
step.
12. Remove all the liquid and let the pellet dry at room temperature for 15
min.
13. Add 100 µL of TE buffer to the dried pellet, tap the tube to disperse
the tissue and incubate at 60°C for 10 min.
14. After centrifugation at 12 000–14 000 rpm for 30 s, transfer the solu-
tion containing DNA to a new tube for further use.
15. The yield of DNA was quantified by DyNA Quant 200 (Amersham
Pharmacia Biotech).

GAGAGT-3′; the Nucleic Acid-Protein out to test the DNA samples extracted
Service Unit, Biotechnology Laborato- with our protocol and with some other
ry University of British Columbia, Van- methods, such as CTAB (1), modified
couver, BC, Canada) and template CTAB (4) and modified SDS (6). The
DNA of B. gymnorrhiza, were carried amplification conditions adopted in this

Figure 2. Electrophoretic analysis (1.5% aga-

× TBE buffer) of PCR-amplified
rose gel in 0.5×
ISSR products using UBC primer no. 807 (5′′-
Figure 1. Electrophoretic analysis (0.8% AGAGAGAGAGAGAGAGT-3′′) and template
agarose gel in 0.5× × TBE buffer) of isolated DNA of B. gymnorrhiza. Lanes 1–4, isolated
DNA. Lane 3, B. gymnorrhiza; lane 4, I. batatas; with CTAB (1); lanes 5–8, modified CTAB (4);
lane 5, S. caseolaris; lane 6, S. alba; lane 7, A. lanes 9–12, modified SDS (6); lanes 13–16, our
formosana; and lane 8, A. tricolor. Lane 1, λ CTAB with silica method; lane 17, molecular
DNA; lane 2, λ DNA digested with HindIII. weight markers (100 bp ladder of DNA).

Vol. 28, No. 3 (2000) BioTechniques 433

Benchmarks
study were as described in Reference 3. DNA will be low. A pH of 6.5 is opti- mosana, A. zerumbet, Boesenborgia fal-
Amplifications were carried out in 1.5 mal for DNA binding. Chloroform ex- lax, Hedychium flarum, Globba bathei,
mM MgCl2, 2% formamide, 200 nM traction is necessary in the procedure Curcuma kwangsiensis, Ethingera yun-
primer, 1 U Taq DNA polymerase and because chloroform can extract CTAB, nanensis and Amomum villosum.
10 ng of genomic DNA per 20 µL reac- which reacts with the chaotropic salt in In summary, our minipreparation
tion. The following PCR cycle profile the supernatant. The pH of elution procedure combines the advantages of
was used: 1 cycle at 94°C for 5 min fol- buffer also affects the yield of DNA, CTAB and silica-cleaning methods. It
lowed by 45 cycles at 94°C for 45 s, and a pH of 8.0–8.5 is adequate for a is rapid (150 samples per day), phenol-
50.5°C for 45 s, 72°C for 1.5 min and a high elution rate. free and yields high-purity DNA that is
final 7 min extension at 72°C. The am- With optimal binding conditions, suitable for most plant molecular ge-
plified products were electrophoresed typical yield was 3–4 µg DNA per 100 netic studies, including cloning experi-
on 1.5% agarose gels and detected by mg fresh leaves of B. gymnorrhiza. ments and DNA sequencing.
staining with ethidium bromide. This is enough DNA for at least 200
The yield of DNA with our protocol RAPD PCRs. Absorbence ratios of
depends on the rate of binding and elu- DNA at A260/A280 were between 1.9 REFERENCES
tion of DNA to and from silica matrix. and 2.0. The length of isolated DNA
Three chaotropic salt solutions (guani- was around 50 kb in the species tested 1.Doyle, J.J. and J.L. Doyle. 1987. A rapid
DNA isolation procedure for small quantities
dine thiocyanate, sodium iodide and (Figure 1). The DNA was free of conta- of fresh leaf tissue. Phytochemical Bulletin
sodium perchlorate) were tested for op- minants interfering with digestion by 19:11-15.
timal yield of DNA. The highest DNA restriction endonucleases (we tested 2.Hoss, M. and S. Paabo. 1993. DNA extrac-
yield (6.4 µg DNA per 100 mg fresh RsaI, HindIII, EcoRI and BamHI, all tion from Pleistocene bones by a silica-based
purification method. Nucleic Acids Res.
leaves) was obtained with 2.0 M guani- requiring different reaction buffer sys- 21:3913-3914.
dine thiocyanate, pH 6.5. tems). PCR amplifications of ISSR 3.Huang, J.C. and M. Sun. Genetic diversity
The pH is the most critical factor for were used to test DNA samples extract- and relationships of sweet potato and its wild
a high yield of DNA. If the pH is higher ed by our protocol and the CTAB (1), relatives in Ipomoea series Batatas (Con-
volvulaceae) as revealed by inter-simple se-
than 7.5 or lower than 4.5, the yield of modified CTAB (4) and modified SDS quence repeat (ISSR) and restriction analysis
(6) methods. DNA extracted by our of chloroplast DNA. Theor. Appl. Genet. (In
protocol was as good as that from the press).
more complicated and time-consuming 4.Rogers, S.O. and A.J. Bendich. 1985. Ex-
SDS method (Figure 2). No amplifica- traction of DNA from milligram amounts of
fresh, herbarium, and mummified plant tis-
tions occurred with DNA extracted by sues. Plant Mol. Bio. 5:69-76.
the CTAB method. Larger DNA frag- 5.Vogelstein, B. and D. Gillespie. 1979. Prepar-
ments were not amplified or amplified ative and analytical purification of DNA from
in low quantity from the modified agarose. Proc. Natl. Acad. Sci. USA 76:615-
619.
CTAB method, leading to weak or 6.Ziegenhagen, B., P. Guillemaut and F.
missing bands in the gel. This indicated Scholz. 1993. A procedure for mini-prepara-
that DNA isolated by the two CTAB tion of genomic DNA from needles of silver
methods might contain substances that fir (Abies alba Mill.). Plant Mol. Biol. Re-
interfere with the PCR. porter11:117-121.
Our method has advantages over oth-
er silica-based isolation methods (2) be- This work was supported in part by a
cause of its rapidity (DNA can be ob- grant from Hong Kong Research Grants
tained within 1.5 h) and high DNA Council to M.S. Address correspondence to
yield. Based on our protocol, we have Dr. Mei Sun, Department of Zoology, Uni-
succeeded in isolating DNA from 35 re- versity of Hong Kong, Pokfulam Road,
calcitrant species belonging to six plant Hong Kong, People’s Republic of China. In-
families: Sonneratiaceae: S. alba, S. ternet: [email protected]
apetala, S. caseolaris, S. hainanensis, S.
ovata, S. paracaseolaris, Duabanga Received 17 September 1999; accept-
grandiflora; Rhizophoraceae: B. gym- ed 16 December 1999.
norrhiza, Ceriops tagal; Myrsinaceae:
Aegiceras corniculatum; Verenaceae:
Avicennia marina; Convolvulaceae: I. Junchao Huang, Xuejun Ge
batatas, I. cynanchifolia, I. cordatotrilo- and Mei Sun
ba, I. grandifolia, I. leucantha, I. ramo- University of Hong Kong
sissima, I. tiliacea, I. triloba, I. trifida, I. Hong Kong, P.R. China
umbraticola, I. lacunosa, I. tabascana,
I. tenisissima, I. alba, I. cairica, I. aris-
tolochiifolia; Zingiberaceae: A. for-

Vol. 28, No. 3 (2000)