Molecular Biology Week 2. Flashcards
(51 cards)
Name the five steps involved in DNA isolation.
Release Disrupt and prep cell free extract Remove Debris from extract Purify DNA from extract Measure quantity and quality of DNA.
What is an example of a specimen with nucleated cells?
A common example is WBCs from blood or bone marrow.
Name the long labour intensive method used to separate WBC from the plasma and other components and cells?
Differential Density gradient centrifugation. Ficoll Gradient.
Name the more efficient method of separating WBC from plasma and the other components and cells?
Differential Lysis which uses the osmotic fragility of the cells.
Hypotonic buffer = lysis of RBCs before WBCs. Centrifugation… pellet of WBCs.
What state are tissue samples in before DNA isolation?
Fresh or frozen tissues must be dissociated (separated)
Name the three techniques of dissociating a tissue sample.
Grinding frozen in liquid nitrogen.
Homogenising tissue.
Mincing using a scalpel.
How are fixed embedded tissues deparaffinated?
Through using xylene or less toxic xylene alternatives. Then tissue must be rehydrated.
What can be prepared from the released cells of a tissue sample?
Free cell extract, cells must be lysed.
What do some bacteria and fungi have in common that must be broken down before a cell extract is obtained?
Tough cell walls.
Name the 4 steps involved in obtaining a cell extract from a microorganism sample.
Culture is grown and harvested.
Cells are removed and broken down to produce extract
DNA is purified.
DNA is concentrated.
Name the three conditions which determine cells become a cell free extract.
The cell type (prokaryotic or eukaryotic)
Presence of a cell wall
Composition of the wall.
Name 4 methods involved in preparing cells to become a cell free extract/methods of cell wall and membrane disruption.
Naturally using a gentle buffer.
Enzymes
Grinding or vigorously mixing with beads/magnets
Treatment with detergents and bases such as NaOH
Name three methods of purifying cell free extract to becoming purified DNA.
Organic
Inorganic
Solid Phase Extraction
When purifying DNA which two methods are used in both inorganic and organic techniques. Which one method is unique to inorganic technique?
High salt
Low pH
Inorganic; Mix of chloroform and phenol
What does high salt, low pH and a mix of chloro and phenol do to the contaminants of DNA
Dissolves the hydrophobic contaminants such as lipids and lipoproteins and collects cell debris and strips away most DNA associated proteins.
What is used to degrade RNA?
RNase.
What does chloroform and phenol develop?
Develops a biphasic layer emulsion. After centrifugation; hydrophobic layer at bottom, hydrophilic on top with a white interface in the middle.
What is the white interface in the middle of the biphasic emulsion made up of?
Amphipathic components. Coagulated proteins.
What charge is DNA and RNA and what is its nature towards water?
DNA and RNA is hydrophilic and is negatively charged.
What happens to the test tube with the biphasic layer and the white interface of amphiphathic cells?
Upper hydrophilic layer is collected, DNa is precipitates using ethanol and a high conc of salt.
The Precipitate is collected, rinsed to remove the salt.
DNA is then dissolved in a buffer.
What is the safety issue with phenol?
“salting out” can be dangerous.
How does the inorganic isolation method differ from the organic method in terms of separating the DNA in the solution.
Use of low pH and high salt to selectively precipitate proteins, separating DNA in the solution. Sodium Acetate.
How is DNA collected after being separated in the solution?
It is precipitated in alcohol and then is pelleted and resuspended in buffer.
What is used in solid phase isolation which is different to the two other methods?
DNA extraction is done using solid matrices.
