LECTURE 12 Flashcards
4 steps of DNA isolation
- cell lysis
- DNA purification from cell extract
- concentrate DNA
- measurement of DNA purity and conc
biological methods of cell lysis
using enzymes to disrupt cell membrane
what enzyme used to disrupt cell membrane - plants?
cellulase
what enzyme used to disrupt cell membrane - bacteria?
lysozyme
enzyme used to disrupt cell membrane - eukaryotes?
sappanin
2 physical methods of cell membrane disruption
osmotic pressure
freeze-thaw
mechanical methods of cell membrane disruption
grinding
what is method of DNA purification - phenol-chloroform ?
lysed cells are mixed with equal volumes phenol: chloroform
centrifuge left with 3 layers
1. phenol/chloroform
2. interface precipitated proteins
3. aqueous layer of DNA
DNA purification kits
a column that contains a silica membrane that binds to DNA in high salt conc
benefits of using commercial kits over phenol:choloform
less hazardous
more pure DNA
less time consuming
benefits of using Phenol:chloroform over commercial kits
cheaper
restriction endonucleases
Molecular scissors that cut at specific known sites in DNA.
why are restriction endonucelases used?
- to make recombinant DNA molecules
- to cut DNA into defined fragments
where do restriction endonucelases act?
phosphodiester bond within a nucleotide
how do restriction enzymes cut DNA?
-make one cut in each sugar phosphate backbone of double helix
-between 3’ O and P
