Download Isolation of Escherichia coli Chromosomal DNA - RIT

Experiment 1
Isolation of Escherichia coli
Chromosomal DNA
In this lab we will isolate E. coli chromosomal DNA for use in several experiments throughout the semester.
The basic protocol involves lysing the cells with lysozyme and SDS. Peptidoglycan strips away the
peptidoglycan in the cell wall, leaving only the lipopolysaccharide (LPS) layer to hold the cells together.
Such cells are referred to as protoplasts or spheroplasts. Final lysis occurs when sodium dodecyl sulfate
(SDS), a mild detergent, is used to dissolve the LPS. The lysate is then treated with proteinase K and RNase
A to degrade proteins and RNA, respectively. Next, the lysate is treated with phenol/chloroform to remove
all soluble proteins. Finally, the DNA will be precipitated with ethanol and recovered by spooling it on to a
glass hook. We will analyze the DNA by UV spectroscopy in Experiment 2.
Materials 250 ml overnight E. coli culture
lysozyme solution (5 mg/ml)
proteinase K solution (100 mg/ml)
RNase A solution (10 mg/ml)
Procedure
10% SDS
phenol/chloroform
ice-cold ethanol
1.
Obtain a 250 ml flask of cell culture.
2.
Centrifuge the cells in 250 ml bottles for 10 minutes at 7000 rpm.
3.
Resuspend the cells in 10 ml of TE buffer and transfer to a 50 ml
orange-capped conical centrifuge tube.
4.
Add 0.5 ml of lysozyme and incubate at 37O for 30 minutes.
5.
Incubate for 10 minutes at 65O to inactivate cellular nucleases.
6.
Add 100 ul of proteinase K and 20 ul of RNase A to your sample.
Incubate at 37O for 30 minutes.
7.
Add 0.5 ml 10% SDS and rock gently.
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Experiment 3
8.
Add 5 ml phenol/chloroform. Rock for 10 minutes and then spin
briefly in a clinical centrifuge.
Phenol/chloroform is immiscible in water and centrifugation will
produce two phases. The lower organic phase contains dissolved
protein. The upper aqueous phase contains DNA. Carefully
transfer the aqueous phase to a fresh tube. The organic phase is
discarded in the organic waste bottle.
CAUTION: PHENOL/CHLOROFORM IS CAUSTIC
YOU MUST WEAR SAFETY GLASSES AND GLOVES!!
9.
Repeat the phenol/chloroform treatment.
10.
Transfer the aqueous layer to a fresh tube and gently add 5 ml of
ice-cold ethanol. The ethanol will form a layer above your DNA
sample, and you should see some DNA precipitate at the interface.
11.
Take several long-tipped Pasteur pipettes and heat the ends in a
flame to form a hook. You will probably want four.
12.
Spool the DNA out of the tube by inserting the hook down into the
DNA layer and then drawing it up while gently swirling. You
should see viscous DNA clinging to the hook. Set the hook aside
and allow the DNA to dry.
13.
When you have spooled out all the DNA wash each hook three
times in cold ethanol. Washes are performed by swirling each
hook in a fresh tube of ethanol.
13.
When the hooks are dry, CAREFULLY snap off the ends of each
into a 15 ml orange-capped centrifuge tube. Add 3 ml of TE buffer
and allow the samples to shake on a rocker platform until next lab.
14.
At the next lab, transfer the dissolved DNA to a fresh tube.
TE Buffer
10 mM Tris, pH7.6
1 mM EDTA
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