DNA (gene) Technology

Question 1

Extraction of DNA
1. Releasing Cell Contents

Answer 1

Finely chopped onion, to which a little salt and detergent added is homogenised using mortar and pestle

Homogenisation disrupts cell walls & detergent causes cell membranes to rupture - cell contents released in solution

Extract filtered - giving filtrate containing soluble proteins and DNA

Question 2

Extraction of DNA
2. Enzymic Breakdown of Proteins

Answer 2

Filtrate incubated with protease to break down the protein framework of the chromosomes and free the DNA

Question 3

Extraction of DNA
3. Precipitation of DNA

Answer 3

Ice cold ethanol added down the side of beaker - floats ontop of filtrate

In presence of Na and Cl ions - ethanol causes the DNA mol to coalesce and precipitate out of sol

Ppt DNA fibres form at the water-ethanol interface

Can be drawn out on a rotated, fine glass rod

Question 4

Extraction of DNA
4. Washing and Resuspending the DNA

Answer 4

Ppt DNA is ‘washed’ with a 70% ethanol solution to remove salts and other water-soluble impurities

Clean DNA then resuspended in a buffer to ensure stability

Question 5

Gel Electrophoresis of DNA

Answer 5

Agarose gel tray prep with wells in the gel at one end

Added to electrophoresis apparatus with wells in end placed towards cathode (-ve) end

Gel covered with buffer

Sample of extracted DNA placed in well

Apparatus connected to electrical supply (80-150V) and subsequent electrical field DNA mol migrate towards anode (+) end

Resultant migrant DNA detected by staining w/ methylene blue

DNA treated with Restriction Endonuclease Enzyme - frag produced will be seperated by gel elec - smaller fragments migrate further in electrical field

Question 7

Why are DNA negative

Answer 7

Presence of phosphate