Comparison of Three CTAB Buffers for Plant DNA Isolation

DNA isolation from plants is traditionally done using hexadecyltrimethylammonium bromide (CTAB) in the extraction buffer, which binds to polysaccharides and makes them insoluble. CTAB reduces polysaccharide contamination that can cause issues with DNA in downstream applications. Also an issue is polyphenol oxidase that polymerizes monophenols following homogenization. The resulting polyphenols, like polysaccharides, can also hinder downstream manipulations of DNA. A good homogenization buffer will include additives such as PVP (polyvinyl pyrrolidone) to remove polyphenols. CTAB extraction buffers should eliminate these issues, making the isolation of high quality DNA considerably easier.

This study makes a comparison between three different CTAB buffers. These included a homegrown CTAB buffer (2% CTAB, 1% PVP, 20 mM EDTA, 100 mM Tris HCl, 1.4 M NaCl), a commercial CTAB formula (G-Bioscience, cat# 786-565), and the OPS Diagnostics CTAB Extraction Buffer (CEB 500-02, CEB 125-01). The buffers were used to isolate DNA from two week old corn plants.

Materials

• Corn leaf tissue
• Homegrown buffer (2% cetyl trimethylammonium bromide, 1% polyvinyl pyrrolidone, 1.4 M NaCl, 0.1 M Tris HCl, 20 mM EDTA)
• CTAB Extraction Buffer (CEB 125-01,CEB 500-02)
• Commercial Buffer (G-Bioscience)
• RNase (RNase A 10 mg/ml in water)
• Chloroform/Isoamyl alcohol (24:1)
• Isopropanol (2-propanol)
• 70% ethanol (Ethyl alcohol)
• TE buffer (10 mM Tris, pH 8, 1 mM EDTA)

Protocol

1. Liquid nitrogen was used to chill a mortar and pestle and pulverize leaf tissue. Frozen powdered tissue (100 mg) was weighed into microfuge tubes.
2. CTAB buffer (500 µl) was added to each tube.
3. The sample was incubated at 60°C for 30 minutes.
4. The sample was centrifuged at 10,000 x g for 5 minutes. The supernatant was transferred to a new tube.
5. RNase A Solution (5 µl) was added  to the supernatant and incubated at room temperature for 15 minutes
6. An equal volume of chloroform/isoamyl alcohol was added to the solution and mixed by vortexing, and then centrifuged for 1 minute at 13,000 x g. The upper aqueous phase was transferred to a clean microcentrifuge tube.
7. Step 6 was repeated until the upper aqueous layer was clear.
8. Isopropanol (0.7 volumes) was added and incubated at -20°C for 15 minutes.
9. The tubes were centrifuged at 13,000 x g for 5 minutes to pellet DNA.
10. The liquid was carefully decanted and the pellet was washed with ice cold 70% ethanol. This step was done twice.
11. The pellet was placed into the SpeedVac with the caps open. The SpeedVac was run on medium heat for about 90 seconds.
12. The dry pellet was resuspended the pellet in 100 µl of TE.

Results/Discussion

CTAB Extraction Buffer (OPS Diagnostics) yielded a higher concentration of DNA followed by the Commercial Buffer, and then the homegrown buffer.  When considering purity, the CTAB Extraction Buffer performed better then commercial and Homegrown buffers for plant DNA extraction (Table 1).

Table 1. Summary of yields and purities of DNA.

Sample	Yield (ng)	260/280	260/230
Homegrown Buffer	7,721	1.93	1.65
Commerical Buffer	6,470	1.94	1.84
CTAB Extraction Buffer	19,141	1.94	1.84

The DNA samples were scanned using a DeNovix spectrophotomter. The resulting scans are illustrated in Fig 1. 

Figure 1. Scans of the DNA preparations from corn using different CTAB buffers. The G-Bioscience buffer is the black line, the Homegrown is the green line, and the CTAB Extraction Buffer is shown in blue.The result from this comparison shows that although CTAB and other additives are important for DNA isolation, other aspects of the buffer formulation are critical. The OPS Diagnostics CTAB Extraction Buffer was analyzed and adjusted to provide higher DNA yields and purity.