MB215 Recombinant Tech

Group:

Experiment 3: Small Scale Plasmid DNA Isolation

Experiment 4: Extraction of Plasmid DNA using commercial Kit and Restriction
Enzyme Digestion

Introduction:
Plasmid DNA can be isolated from bacterial cells and separated from the chromosomal DNA by
several techniques. This is due to its compact size and close circular and superhelical nature.
Chromosomal DNA can be pelleted by centrifugation following cell lysis leaving the plasmid DNA in
solution. Purification requires the removal of protein and isolation of the DNA by cesium chloride
gradient.

The plasmid containing strain is grown to late stationary phase in “plasmid brothy”. Plasmid broth is a
very rich medium that allows cells to grow to a very high density. In addition, the high concentration
of yeast extract promotes the amplification of plasmids yielding a large copy number of plasmids per
cell. The cells are treated with EDTA which chelates divalent cations, weakening the outer membrane.
(Most procedures include lysozyme to degrade the cell wall peptidoglycan, but the yield of DNA is
just as high without lysozyme.) The cells are then lysed with a solution of SDS and NaOH. In addition
to disrupting the cytoplasmic membrane, the SDS denatures much of the cell protein. The NaOH
causes dsDNA to denature because of the high pH. The denatured ssDNA can reanneal when the pH
is lowered by addition of ammonium acetate. However, the rate of reassociation is proportional to the
length of the DNA. Since the plasmid DNA is small and intertwined, it renneals much faster than the
chromosomal DNA. When the mixture is centrifuged, the reannealed plasmid DNA remains in
solution but the large aggreagates of denatured chromosomal DNA, RNA, and protein are pelleted.
The plasmid DNA is then precipitated with isopropanol. The purified DNA is usually stored in a Tris
buffer containing 1mM EDTA (TE). The EDTA chelates heavy metals that could cause nicking of the
DNA and divalent cations that are required by any contaminating nucleases. The DNA can be stored
at -20ºC or 4ºC. (It was previously thought that storage at -20ºC induces nicks in DNA but recent
studies by BRL suggest that even after many freeze-thaw cycles the number of nicks accumulated in
supercoiled DNA were negligible. The main advantage to storing DNA at 4ºC is that you do not have
to wait for it to thaw.

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Extraction of the pBluescript plasmid DNA from the bacteria cells will be carried out using two
methods: a) Manual alkaline lysis method and b) Wizard® Plus SV Minipreps DNA Purification
System (Experiment 4).

Objectives:

Methods and material: Refer to manual

Results

Manual Kit
A260:

A280:

Ratio A260/280:

Plasmid DNA
concentration:

Comment on the steps, ratio and DNA concentration: (any contamination)

Discussion:

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Question:
1. What is the advantage of using conventional method and kit method in isolating
plasmid DNA?

2. What are the functions of restriction enzymes?

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Conclusion:

Suggestion (if any)

References (if any)

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