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Restriction Digestion for DNA
Restriction enzymes are
naturally occurring endonucleases that recognize a specific palindromic
sequence (4-12 bp) and cut the double strand phosphate backbone of DNA within or near the
recognition sequence. The cuts may be opposite each other on the DNA backbone or
staggered. Opposite cuts produce blunt ends, while staggered cuts produce sticky ends. For example,
EcoRI recognizes the
palindrome sequence, GAATTC (note the reverse complement of this sequence is
GAATTC), and
cleaves the DNA 's phosphate backbone between the G and the A on both strands generating
staggered ends that are complementary to each other: (and see another figure
below)
Because restriction endonucleases
recognize specific sequences and cut DNA to produce sticky ends, they are useful for gene
splicing manipulations.
Another enzyme Eco RV creates blunt ends:
- Both sticky and blunt ends may be rejoined by another
DNA modifying enzyme DNA ligase. Restriction enzymes are named after
the organism they are isolated from and usually the temporal order
of their isolation. For example Eco RV is the fifth reported from E.
coli. Try finding the origin of the name for the restriction enzyme
Aat II in a supplier's catalog: Stratagene.
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I. Restriction Digestion of Lambda DNA
Identify and organize the following
reagents on ice. Then, very carefully pipette each in order into a 1.5 ml
Eppendorf tube.
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Lambda DNA (0.1 µg/µl)
10x buffer
MilliQ H2O
EcoRI (enzyme) |
5.0 µl
2.5 µl
16.5
µl
1.0 µl |
| Total volume of the
reaction is: |
25 µl |
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Incubate the reactions at 370C
for 1 hr. |
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Add 2.5ul loading dye and incubate
for another 15 minutes. |
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Store the samples at -200C,
if necessary. |
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Load 20 µl of the digestion mixture on to
a minigel. |
II. Gel Electrophoresis
go on to the gel
electrophoresis page 
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Materials and recipes 
