PAG - Extraction of DNA Flashcards
Plant cell walls -
- need to be broken open through frictional force
Breaking cells and disrupting nuclear membrane -
- releases DNA
Using enzymes -
- denatures histones associated with DNA
histones
proteins associated with DNA
DNA is made less soluble -
- by adding salt
Once insoluble in the water -
- precipitate DNA using an organic solvent
what fruit are we using
strawberry
first step-
- crush strawberry with DNA extraction buffer using a pestle and mortar
- DNA is less likely to clump together
what is the DNA extraction buffer
- detergent and salt
what does the detergent do -
- disrupts the phospholipid bilayer of cell membranes and nuclear membranes releasing DNA
what does the salt do -
- neutralises charges of sugar phosphate backbone, reducing the solubility of DNA in water/cytoplasm
after DNA had been crushed, and salt and detergent is added, what is next
- strain into a beaker
- use tea strain and muslin
- this is to remove debris and membrane fragments, leaving just DNA and its histones
after the DNA has been filtered, what happens
- add 1cm3 protease enzyme obtained from pineapple juice
protease enzyme -
- digests histones, leaving just DNA
- allowing DNA to uncoil
after adding protease, we pour the filtrate into a boiling tube, what next ?
- add 5cm3 of ice cold ethanol
why do we add ice cold ethanol
- to slow down the enzymes which break down DNA
how do we add ethanol to the filtrate
- tip boiling tube to 45 degrees and slowly roll ethanol down inside the boiling tube so it forms a layer on top of the filtrate
how does the ethanol form a layer on top of the filtrate
- it is less dense
- due to the salt in the buffer making DNA insoluble so a white precipitate forms
after adding the ethanol we let the filtrate stand for 5 minutes, what forms
- a white precipitate
- this happens where a DNA layer buffer meets layer of ethanol
how do we extract the white precipitate
- using an inoculating loop
what may scientists do with the extracted DNA
observe it under a microscope
Students are extracting DNA from white blood cells, suggest why they do not need to crush up the wbc with a pestle and mortar
- they do not have cell walls to break open
Students wanted to purify DNA from leek cells by extracting its DNA, suggest why using a pineapple juice as a source of protease may not be appropriate
- pineapple juice contains DNA
- pH may be too low
what is the name of the process when a white layer of DNA forms between the sample and the ethanol
precipitation
A student tried to extract some DNA from a crushed banana at home. DNA dissolves in water but the
student realised that they needed to add something to break open the nuclear envelope to release the DNA.
Suggest a suitable substance the student could use to release the DNA, and explain why it should work
- detergent
- works as an emulsifier / attracts
phospholipid molecules and
water molecules - it will break up the plasma /
nuclear membranes
