Transcript Plasmid Isolation

Plasmid Isolation
RET Summer 2007
Overall Picture
Plasmid Isolation
Remove plasmid pBS 60.6 from DHa E. coli
Restriction Enzymes
• Each restriction enzyme cuts at different sites
• The cut can leave either blunt or sticky ends
• If the plasmid and gene to be inserted into the plasmid
are cut with the same restriction enzyme, the sticky
ends have overlapping base pairs and can anneal with
DNA ligase
• But, the cut ends can anneal to themselves or the cut
vector can reanneal to itself
• Can prevent vector religation by using two restriction
enzymes at opposite ends of the insert and plasmid
Plasmid Isolation
• First, separate cells from growth medium
• Lyse host cells to release the plasmid DNA but
not the genomic DNA
– usually done in alkaline solution and SDS
• Neutralize with acid to allow genomic DNA to
renature and aggregate
– Large DNA and proteins bound to SDS will ppt
Isolating Recombinant Plasmids
•Cells are resuspended in buffer
(P1) containing Tris, EDTA, and
RNase.
•EDTA stabilizes the cell
membrane by binding divalent
cations (Mg++ and Ca++).
•RNase destroys the cell’s RNA.
NOTE: DO NOT VORTEX!
Source: DNA Science: The First Course in Recombinant DNA Technology
Isolating Recombinant Plasmids
• The resuspended cells are
then treated with a SDS and
sodium hydroxide (P2).
• SDS dissolves the
phospholipid and protein
components of the cell
membrane.
• The cell membrane lyses,
releasing the cell contents.
NOTE: DO NOT VORTEX!
Source: DNA Science: The First Course in Recombinant DNA Technology
Isolating Recombinant Plasmids
• Sodium hydroxide
denatures both plasmid and
chromosomal DNA into
single strands.
• Chromosomal DNA
separates completely into
individual strands.
• The single-stranded
plasmid loops remain
linked together like
interlocked rings.
Source: DNA Science: The First Course in Recombinant DNA Technology
Isolating Recombinant Plasmids
• Acidic potassium acetate (N3)
forms an SDS/lipid/protein ppt.
• Acetic acid neutralizes the
NaOH.
• At neutral pH, genomic DNA
renatures and is trapped in the
SDS/lipid/protein precipitate.
• The plasmid DNA renatures into
double-stranded molecules that
remain in solution.
Source: DNA Science: The First Course in Recombinant DNA Technology
NOTE: DO NOT VORTEX!
Isolating Recombinant Plasmids
• The precipitate is pelleted by
centrifugation.
• The plasmid DNA and small
RNA molecules remain in the
supernatant.
• The supernatant will be
transferred to a clean tube.
The pellet with the cellular
debris is discarded.
Source: DNA Science: The First Course in Recombinant DNA Technology
Isolating Recominant Plasmids
• Load supernatant with
plasmids onto spin columns.
• The pellet is washed with
EtOH (PE), causing the
plasmid DNA to ppt.
• The plasmid DNA binds the
resin in the spin column under
high salt conditions.
• Finally, the purified plasmid
DNA is eluted in water.
Quantifying Plasmid DNA
Quantify DNA using UV absorbance
– DNA UV absorbance peaks at 260 nm
– protein UV absorbance peaks at 230 nm
(peptide bond) and 280 nm (aromatic amino
acids)
– The ratio of the absorbance at 260 nm/280 nm
is a measure of the purity of a DNA sample; it
should be between 1.65 and 1.85.
Analyzing Recombinant Plasmids
• Purified plasmid DNA has
now been isolated from
transformed bacterial
cells.
• After quantifying DNA
yields, restriction analysis
is performed on the
purified plasmid DNA.
B
Lac Z gene
Ampicillin
resistance
gene
B
puc plasmid vector
Origin of replication
Analyzing the Plasmids
•Samples of the miniprep
(purified) plasmid are
digested with restriction
enzymes.
•You will digest your
isolated plasmid with 2
restriction enzymes:
BamHI and SacI.
•The resulting restriction
fragments are separated by
gel electrophoresis.
Source: Biotechnology: Demystifying the Concept
Digesting the Plasmids
You will have 4 tubes for the restriction digestion. Each tube should
contain 0.5 mg of plasmid DNA. You will have to calculate the
appropriate volume you need from your sample to get 0.5 mg of
DNA. Total final volume for each tube is 10 mL, so adjust the
amount of H2O accordingly.
DNA
mL
BamHI
mL
SacI
mL
Buffer
mL
1
Calc*
1
1
1 - Multi
dH2O
mL
10-3-calc
2
Calc*
1
0
1–J
10-2-calc
3
Calc*
0
1
1–E
10-2-calc
4
Calc*
0
0
1- Multi 10-1-calc
Analyzing Recombinant Plasmids
•After digestion, the
restriction fragments are
separated by gel
electrophoresis.
•SyberSafe fluorescently
labels the DNA, which can
then be visualized under
UV light.
•The banding pattern from
the restriction fragments
provide a genetic
“fingerprint” of the
plasmid and gene insert.