DNA extraction & gels.ppt


14 Δεκ 2012 (πριν από 5 χρόνια και 7 μήνες)

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After discovering DNA was the carrier of
genetic info, it became apparent that control
over its mechanisms would be essential

We have ways of isolating DNA, determining
the order of its nucleotides, and can even
insert of piece of DNA from one organism to

To get at DNA, we have to remove the two
membranes that protect it

Cell membrane and nuclear envelope

These are made of phospholipid bilayers

Detergents are used to dissolve the lipids to
expose the genetic material

Next, a protease is used to dissolve unwanted

Sodium acetate further precipitates the
remaining protein

Next, the sample is centrifuged, and the
protein can be removed

DNA can be precipitated by the addition of
cold ethanol

This both precipitates the DNA and washes the salt

Finally, the solution is centrifuged, and the
pellet of DNA is removed

The DNA extracted is often very long

Too long to be useful in many cases

Restriction enzymes are used to cut the DNA

However, it is not a random cut site, rather a
very specific sequence

There are many different enzymes (~1000),
many with their own unique sequence they
attach to

They do not cleave the DNA straight down,
instead the cut the bond between adjacent

The uneven cutting creates what is known as
“sticky ends”

They are “sticky” because they are single

DNA is normally doubled stranded, and naturally
anneals (reforms hydrogen bonds) into double

There is a random number of cut sites on
every strand of DNA

But, in the end, the sample is cut into smaller

These smaller segments can be run through a

The gel is a semi
solid, porous medium that
DNA can move through

Electrodes are set at either end to make one
positive, the other negative

DNA is slightly negative, and will be attracted
to the positive end

The smaller samples can move through the
gel faster, and will make it farther down

Each band represents a strand of DNA, of a
specific length

The bands furthest down are the smallest

Gels are normally run to compare a sample of
DNA to a known sample, cut with the same

If the bands line up, it is likely that the
samples are the same

This is very useful in DNA fingerprinting