Download Lab 6 DNA ISOLN

CHROMOSOMAL & PLASMID
DNA ISOLATION FROM
BACTERIAL CELLS

PLASMIDS
Small extrachromosomal circular dsDNA molecules
Not crucial for bacterial survival
Confers bacteria w/ selective advantage
» Synthesis of toxins - pathogenicity
» Antibiotic resistance - Resistance-transfer factors
RTFs
» Synthesis of the F pilus
» Metal tolerance
» Novel enzymatic activities
» Synthesis of Adhesins
Utilization as cloning vehicles in biotechnology
BACTERIAL PLASMIDS
CHROMOSOMAL DNA
ISOLATION
Two Methods:
1. Spooling
2. Extraction
Spooling
DNA Extraction

The basic steps of Chromosomal DNA isolation
are:
1.
Disruption of the cellular structure
Separation of the soluble DNA from cell debris
and other insoluble material and
Purification of the DNA of interest from soluble
proteins and other nucleic acids
2.
3.
Culture centrifugation
PLASMID DNA ISOLATION
FROM BACTERIAL CELLS

For the selective isolation of plasmid DNA,
cells are exposed to NaOH and a strong
detergent (alkaline lysis) to denature de
Chromosomal DNA (i.e. the 2 strands are
separated). An acidic solution of sodium
acetate is then added to neutralize the
solution. At this point, most of the cell
membrane material and the genomic DNA
are spilled out and precipitate .
 The selective precipitation of Genomic DNA
is based in its higher molecular weight.
DNA Isolation Kits





Kits for plasmid DNA purification are commercially
available and provide faster and more accurate
isolation and purification procedures
They are mostly based DNA-binding in columns
after cell lysis and precipitation of insoluble
materials
Advantages: Faster and higher yields of DNA
We are going to use a Commercial Kit for plasmid
DNA isolation from E.coli pDSRed2, containing the
red fluorescent gene
All samples containing DNA should be kept on ice
during the isolation process