The CTAB method is biochemically simple, easy to learn, and relatively cheap to perform. However, in practice, the protocol has several drawbacks: it’s lengthy, tedious, and low throughput, with many steps that require careful handling, exposure to hazardous chemicals, and several other technical considerations.
Timing Your DNA Extractions
From grinding with a mortar and pestle to resuspending sticky DNA pellets, the full CTAB protocol can take approximately two hours to process a small number of samples. There are also more than 20 steps in the protocol and as the number of samples increases, the amount of time needed to complete DNA extraction increases substantially. Plan your day carefully and set aside the proper amount of time to complete the entire protocol.
Working With Hazardous Materials
Caution must be taken when working with liquid nitrogen for the first grinding step as it can rapidly freeze skin tissue and cause cold burns even with short exposure. In addition, working with phenol and chloroform is also a biosafety hazard: Phenol can cause chemical burns and chloroform is a potential carcinogen.6,7 For many food testing labs, the use of these toxic chemicals is a major concern. Use of phenol/chloroform also generates organic waste which requires special storage containers and disposal procedures. Be sure you have the proper safety protocols in place before starting your DNA extractions.
Processing the Plant Tissue
Tissue grinding can vary between samples, leading to significant variation in extraction efficiencies and quality of DNA. To achieve more consistent tissue disruption across samples, you can also use a blender, though this step is still low throughput and time consuming. The more finely your tissue is ground, the more efficient you DNA extraction will be, making this a critical step for successful DNA extraction.
A solution of phenol/chloroform/isoamyl alcohol is used to extract plant DNA from cellular debris and once added and vortexed, the mixture separates into three distinct phases: aqueous, interphase, and organic phase. While removing the aqueous phase and repeating the extraction is time consuming and laborious, it can also be challenging to remove all the aqueous phase, without disturbing the interphase. As a result, you may leave DNA behind or carryover contaminants from the interphase and organic phase, lowering your overall DNA yield and quality.
Several classes of biochemicals from plant tissues – polysaccharides, lipids, polyphenols, and/or other secondary metabolites – can coprecipitate with DNA, which can inhibit downstream applications that rely on thermostable DNA polymerases, such as PCR. The structure and concentration of these compounds can also vary substantially between different plant species, making the development and optimization of a “one size fits all” CTAB protocol difficult.8,9 In addition, phenol and other salts introduced throughout your CTAB protocol can remain, even after extensive ethanol washes. These impurities can also interfere with downstream applications, including PCR and NGS.10