CHAPTER 10/ LAB 10
•
Alkaline Lysis mini-prep to isolate plasmid from bacterial cells
•
DNA replication
•
PCR to amplify large amounts of DNA in a few hours
•
PLASMID
o
Small, circular DNA in prokaryotes
o
Carries non-essential genes
antibodies, metabolizing unusual food
sources, producing toxin
o
Transferred by
Transformation
or
Conjugation (
mating process)
•
Selection: selective media
o
Containing an antibiotic
o
Containing or lacking a specific required nutrient
•
Stringent control: replicates when chromosome does; only 1 copy
•
Relaxed control
: uncoupled with chromosomes
hundreds of copies
•
3 states of plasmids:
o
Supercoiled
: favorable state
o
Coiled
: one coil from supercoiled is broken (nicked backbone); open
circular
o
Linear
: Backbone of both strands of double helix are broken
•
Goal of Plasmid Extraction
:
o
Separate plasmid DNA from chromosomal DNA & protein
o
ALKALINE LYSIS PROCEDURE:
1.
Grow bacteria overnight in Luria Browth and centrifuge
2.
Resuspend in
STE buffer (
NaCl, Tris buffer, EDTA at pH 8)
3.
Centrifuge, keep pellet cells,
PELLET
in
GTE BUFFER
(Tris,
glucose, EDTA)
4.
pH 8
5. ADD
SDS detergent(contains NaOH)
solution (pH = 12)
•
Completely denatures chromosomal DNA
•
Partially denaturing plasmid DNA
6.
Ice for 5 minutes
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7. Add
Potassium Acetate
(pH 5.5) to precipitate cell debris (proteins,
chromosomal DNA)
8.
Vortex and chill for 5 min.
9.
Put in microcentrifuge at 4
o
C for 5 minutes
10.Discard pellet, keep
supernatant
11.Centrifuge again, with 95% ethanol – 3 minutes at 80% full speed
12.Pour supernatant into 25 mL beaker,
KEEP PELLETS
13.Transfer 1mL of 70% ethanol
14.Centrifuge again,

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- Fall '07
- Hanson
- molecular biology, Bacteria, DNA
-
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