Can CTAB be used for animal DNA isolation?

Can CTAB be used for animal DNA isolation?

We modified a low-salt CTAB (MoLSC) extraction protocol to accommodate contaminant-rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle.

How is DNA extracted using CTAB extraction?

One of the most commonly used methods to extract DNA from plants uses the ionic detergent cetyltrimethylammonium bromide (CTAB) to disrupt membranes and a chloroform-isoamyl alcohol mixture that separates contaminants into the organic phase and nucleic acid into the aqueous phase.

What is CTAB DNA extraction?

CTAB (also called hexadecyltrimethylammonium bromide) is a cationic detergent that facilitates the separation of polysaccharides during purification while additives, such as polyvinylpyrrolidone, aid in inactivating polyphenols. CTAB based extraction buffers are widely used when purifying DNA from plant tissues.

How do you extract DNA from animal cells?

Animal Tissue DNA-Extraction & WGA Amplification Protocol

1. Place 20 mg of tissue into a microcentrifuge tube.
2. Digest Tissue: Resuspend the tissue pellet in 180 μL of Lysis Solution T.
3. Add 20 μL of proteinase K, mix by vortexing.
4. Incubate at 55 °C for 2–4 hours or overnight until the tissue is completely lysed.

What is the role of EDTA in DNA extraction?

The EDTA works as a chelating agent in DNA extraction. It chelates the metal ions present in the enzymes, metal ions work as a cofactor to increase the catalytic activities of an enzyme. In DNA or RNA extraction, the use of EDTA readily deactivates DNase or RNase enzymes which digest DNA or RNA, respectively.

Why is chloroform used in DNA extraction?

The main function of chloroform is to protect genomic DNA during a catastrophe. Chloroform increases the efficiency of phenol to denature the protein. Here, chloroform allows proper separation of the organic phase and aqueous phase and keeps DNA protected into the aqueous phase.

How is genomic DNA extracted from plant tissues?

Plant tissue is harvested and placed in tubes or plates. The tissue is homogenized to separate the cells from each other. The plant cells and nuclei are lysed in the presence of an extraction buffer; the buffer contains salt and chemicals to help lyse the plant cells, stabilize the DNA, and reduce degradation.

Why is CTAB used?

Cetyltrimethyl ammonium bromide (CTAB) is a surfactant useful for isolation of DNA from tissues containing high amounts of polysaccharides. Under the high-salt conditions used in this protocol, the CTAB binds the polysaccharides, removing them from the solution.

What is the first step in DNA extraction from animal tissues?

The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.

1. Step 1: Lysis. In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this.
2. Step 2: Precipitation.
3. Step 3: Purification.

How do you extract DNA from a tissue?

Preparing the Tissue Lysate

1. Place the tissue sample into a sterile microcentrifuge tube.
2. Add 1 ml of Lysis Mix (see above) to the tube.
3. Vortex for 10-15 seconds to mix.
4. Incubate the sample overnight at 55° C until lysis is complete.
5. Add 5 µl of RNase A to the lysate.
6. Incubate at room temperature for 5 minutes.

Why is isopropanol used in DNA extraction?

The overall function of salt and ethanol/ isopropanol is to precipitate DNA from the solution. The salts neutralize the negative charge of the negatively charged phosphate in DNA and the isopropanol /ethanol removes the hydration shell of H2O molecules around the phosphate.

Which is better CTAB or Edwards for DNA extraction?

For the CTAB method, the mean DNA yields from individual tissues ranged from 341.7 (±97.6) to 897.2 (±110.7) μg/μl and for the Edwards method, 300.0 (±81.0) to 1558.3 (±337.5) μg/μl. The mean DNA concentrations were higher using the Edwards method from all tissues tested, except mature leafs and petals.

What is the function of a CTAB in DNA isolation?

CTAB based extraction buffers are widely used when purifying DNA from plant tissues. One option for purifying DNA using CTAB exploits that polysaccharides and DNA have different solubilities in CTAB depending on the concentration of sodium chloride.

Can a CTAB be used for chloroform extraction?

CTAB-based protocols tend to work very well, but one significant disadvantage is that chloroform extractions are routinely used to separate organic soluble molecules from the DNA. As chloroform is carcinogenic, many institutions frown upon its use.

What kind of buffer is used for CTAB extraction?

CTAB buffer: 2% cetyl trimethylammonium bromide, 1% polyvinylpyrrolidone, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, or CTAB Extraction Buffer Plant samples can be prepared by cryogenically grinding tissue in a mortar and pestle after chilling in liquid nitrogen. Freeze dried plants can be ground at room temperature.