Isolating DNA: Lecture 11 Flashcards
What are the 3 types of methods for cell lysis?
Biological, Physical, Chemical
4 steps of DNA isolation?
- Cell lysis
- DNA purification from cell extract
- Concentrate DNA
- Measurement of DNA purity and conc
Unwanted components of DNA extract sample?
lipids, proteins, mtDNA, ribosomes
What’s biological cell lysis and examples?
Using enzymes to disrupt cell membranes
Plant- cellulase
Bacteria- lysozyme
Eukaryotic cells- sappanin
What’s physical cell lysis and example?
Using osmotic pressure- when cells put in hypotonic solution cell swells and explode.
Freeze-thaw method: repeated cycle of freezing and thawing rupture cell membranes from ice formation
What are the 3 methods of mechanical lysis?
Pestle and mortar- disrupt plant cells, hard tissue
Vortex- used w/ beads or by itself for small no. cells
Bead mill- used to beat and grind tough samples
What are the 2 methods of DNA purification?
Phenol- chloroform extraction and commercial kit
How does Phenol-chloroform extraction work?
- Lysed cells/tissue mixed with equal volume of phenol-chlor mixture
- Centrifuging gives 2 separate phases because phenol chlor don’t mix with water
- dna concentration- 0.3M sodium acetate &2.5 vol ethanol used to precipitate DNA from salt and sugar to concentrate it.
What do the phases/layers look in the phenol-chloroform extraction?
top aqueous layer = DNA
middle interface = precipitated proteins
bottom= phenol-chlor
How does Commercial kit work?
- Column contains silica membrane which bind to DNA in presence of high salt concentration
- Impurities like salt washed away
- Low salt buffer used to release DNA from membrane and collect it.
points for phenol-chloroform vs commercial kit?
- commercial kit less hazardous
- commercial kit less time consuming
- commercial kit gives purer DNA than phenol-chlor
Techniques for measuring quantity and quality of DNA?
- UV absorbance
- Gel electrophoresis
- Fluorescence dyes
- Capillary electrophoresis
- Diphenylamine method
How to name restriction enzyme?
genus
species
strain
type
How do restriction enzymes make the cut?
Between the 3’ oxygen and phosphate group in sugar phosphate backbone.
In the presence of Mg2+
Hydrolysis
The cut end has the 5’ phosphate group
what is star activity?
When the enzyme cuts where it shouldn’t. Due to low ionic strength, high pH, high glycerol conc, maybe presence of Mg2+.
Reaction conditions different to optimum and chemical/drug intervention affecting specificity
