What are the three basic steps for DNA extraction?
DNA extraction is defined as isolation of DNA from the nucleus of cells. But pure DNA is obtained by separation of DNA from cellular fluid and proteins.
Then this pure DNA can be used for DNA fingerprinting, sequencing, cloning, PCR etc.
The basic steps of DNA extraction are:
1) Lysis :
The first step is lysis in which the cells in the given sample are separated from each other by mechanical or physical disruption.
Mechanical disruption are mostly used in plant cells as they have a tough cell wall. In this method, tissue homogenizer which is like a small blender is used with a mortar and pestle. This helps in cutting of tissue into small pieces.
Physical disruption uses methods such as grinding or vortexing then add into salty solution. The positively charged sodium ions present in salt protect the negatively charged phosphate which is present along the backbone of the DNA. Then add detergent that breaks down the lipids in the cell membrane and nuclei. DNA is isolated because membranes are disrupted.
2) Precipitation :
After lysis, the DNA has been isolated from the nucleus, but it is not pure as it is mixed with mashed cell parts. Precipitation helps in separation of DNA from the cellular debris.
Precipitation uses Na+ ions (sodium) that neutralizes the negative charge phosphate on the DNA molecules and makes them less soluble in water.
Ice-cold alcohol is also used as DNA is soluble in water but insoluble in the presence of salt and alcohol.
3) Purification :
DNA is now separated from the aqueous phase. After that rinse DNA with alcohol to remove remaining unwanted material and cellular debris.