Quiz Time/1

Plant DNA extraction - CTAB method

1. Why is CTAB used in the extraction buffer? What other alternatives can be used other than CTAB in the extraction buffer?

2. Explain the function of beta mercaptoethanol in the extraction buffer.

3.In which step is chloroform used and why?

4. What is the function of isoamyl alcohol?

5. What are immiscible liquids?

1) CTAB is used in the extraction buffer because it simultaneously solubilizes the plant cell wall and lipid membranes of internal organelles and denatures proteins (enzymes). Thus, the DNA is not hydrolyzed during the isolation process and as long as vortexing or vigorous shaking are avoided highly polymerized (i.e., very high intact) genomic DNA is obtained.

We can use SDS (sodium dodecyl sulphate) and SLS (Sodium lauryl sulphate)

2) we use beta mercaptoethanol to stop protein denaturation.

3) Chlorofoam is used in extraction step and it is used to help separate proteins and polysaccharides from nucleic acids in the cell. The purpose of the Chloroform will be to help bind up the complexed proteins and polysaccharides. Chloroform is more dense than water solutions and thus after spinning this solution Chloroform and water will separate into two distinct phases. The lower phase will be Chloroform. This is the phase that proteins and polysaccharides find most chemically attractive. The upper aqueous phase will contain our DNA. With plant tissue there is often more polysaccharides than in animal tissue. If the polysaccharides are not removed many procedures such as restriction enzyme digests will not work appropriately.

4) We use isoamyl alcohol to minimize bubble formation. (foaming)

5) Immiscible liquids are liquids that doesnt not dissolve with each other.

does not*

1.CTAB is the best detergent to use during the extraction of highly polymerized DNA from plant material. ( Dissolves the cell wall, Denatures the protein and Dissolves the lipid bilayer)
- Alternatives ( SDS and PVP)

2. It is a strong reducing agent.
It may also help to denature proteins by breaking disulphide bonds between cysteine residues.