Restriction endonucleases
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Restriction enzymes which are
able to cut double stranded DNA into fragments at specific sequences
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Recognize 4 to 8 base pair
sequences as the recognition site
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Different enzymes would produce
two different kinds of end, sticky and blunt ends
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Enzymes cut sticky ends, such
as EcoRI, are more useful tools for molecular biologist, because it can be
easily joined to other sticky end fragments that produced by the same enzyme,
for example sticky ends usually use to cut DNA fragments for cloning
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SmaI and AluI cut blunt ends
fragments, these enzymes usually use in body for protection of the original DNA
from virus.
Gel electrophoresis
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Separate DNA fragments
according to size.
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longer the fragment, slower the
movement(migration)
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DNA fragments are negative
charges, the gel electrophoresis use this property to induce the fragments to
move through the gel
-
after the process is complete,
these fragments are made visible by staining the gel with ethidium bromide, the
most commonly used stain.
-
It is also commonly to protein
with polyacrylamide gels
Plasmid
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Small circular pieces of double-stranded
DNA molecules in bacteria
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Carry genes that express
protein able to confer antibiotic resistance
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Protect bacteria by carrying
genes for resistance to toxic heavy metals
-
can replicate independently as
long as they have a favorable environment
-
really useful for cloning
because after placed the gene fragment to sticky ends produced by the same
enzyme, the foreign gene will permanently become part of the plasmid
-
replicate many copies of the recombinant
DNA (copy of the original inserted gene)
Transformation
-
introduction of foreign DNA
into a bacteria cell, usually by a plasmid or virus
-
selective plating is a method
of isolate the cells with recombinant DNA
-
will have expected pattern of
bands of colony on the gel, whether it carry a recombinant DNA plasmid or not
-
calcium chloride method is the
classical method of transforming cells
-
electroporators are also used
nowadays
