Download Prepare for gel electrophoresis

GEL ELECTROPHORESIS
FIRST, WHAT……
 Simply put, gel electrophoresis is a technique used to separate
molecules such a DNA, RNA and proteins according to size.
 Requires an electric current as these molecules are negatively charged
 We’ll be looking at separating long strands of DNA into smaller
fragments.
 Can be used to determine someone’s DNA identity (fingerprint), assign paternity, ID
criminals from a crime scene, determine relatedness among individuals, useful in
cataloging endangered species
FIRST, DNA IS EXTRACTED
 The same DNA is found in the nucleus of nearly all of your cells
(mature red blood cells have no nucleus).
 Mitochondrion have a totally different set of genes than what is in the nucleus.You inherit them
from your mother! Many studies use this DNA to determine how closely related different
species of organisms are!
 Nuclear DNA can be extracted from blood, cheek cells, hair
follicles, tooth pulp or mostly any other tissue
 There are quite a few protocols that use a variety of chemicals and
buffers to isolate ONLY DNA and get rid of everything else
RESTRICTION ENZYMES
 Long strands of DNA, usually from those regions that do not code for
anything, are cut into smaller fragments using restriction enzymes
 Each enzyme recognizes different sites to cut
 This results in many fragments of different sizes (number of bases long)
PREPARE FOR GEL ELECTROPHORESIS
After the gel cools
and becomes jellylike, the comb is
removed and is
placed in the
chamber filled with
buffer
Power supply
A compound called
agarose is melted in a
buffer, poured into a mold
and a tooth comb inserted
to create wells
Gel Chamber
with buffer
“RUNNING A GEL”
A loading dye (seen in
blue) is mixed with DNA
sample and loaded into
a well
DNA Ladder (or
size standard) is
also loaded. This
lets allows you to
determine how
long each
fragment is (in
kilobases)
Electrical supply is
turned on…DNA
fragments run through
the gel from the
negative electrodes
toward the positive
The gel is stained and
examined. The
smallest (shortest)
fragments travel the
farthest down the gel
SOME STAINS REQUIRE
ULTRAVIOLET LIGHT TO SEE DNA
BANDS
DNA FINGERPRINTS ARE UNIQUE TO
EACH PERSON
EXAMPLE OF FINGERPRINTS
Out of the 7 suspects, the
bloodstain found at the
scene of the crime belongs
to……..
A person should have ½ of their DNA fragments from
their mother, and ½ from their father.
**Below: known mom, child and 3 alleged fathers
The child received
these fragments
from its mother…
-
…So Alleged Father
#2 is the father of the
child.
..which means
that they must
have received
these from the
father…
…so, which alleged
father has the same
size fragments as
the child at these
positions???
Mom Child AF1 AF2 AF3
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