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Produced by bacteria
as a defense against
bacteriophage and
other foreign DNA
Named according to
the bacteria that
produce them
Restriction sites
5-NNNG-3
3-NNNCTTAAG-5
5-AATTCNNN-3
3-GNNN-5
Sticky Ends
5-NNNG-3
5-GATTCNNN-3
3-NNNCCTAG-5
3-GNNN-5
Sticky Ends
5-NNNA-3
3-NNNTTCGA-5
http://www.phschool.com/science/biology_place/biocoach/red/cleave1.html
http://highered.mheducation.com/olc/dl/120078/bio37.swf
5-AGCTTNNN-3
3-ANNN-5
Sticky Ends
Uses of
plasmids
Genetic
Engineering,
Biotechnology
or more
specifically
Gene cloning
Our experiment
Experimental design:
1. Setup the digestion of a plasmid using three
restriction enzymes: EcoRI, BamHI and HindIII
2. Run samples on an agarose gel electrophoresis
3. Perform analysis of the results and construct a
restriction map
Gel Electrophoresis
Shorter DNA fragments migrate faster across the gel than longer
DNA fragments which migrate slower
To determine the actual size of the fragments we use known
standards (called marker or ladder)
Gel tray will be perpendicular, once the gel has solidified, place the
gel tray parallel with the gel box, pour the running buffer in the gel box
to cover the gel, then slowly remove the comb. Add you samples to
the wells, making note of which well each sample is in.
DNA is negatively
charged so if
electric current is
applied DNA will
migrate towards
the positive pole
Thus, it is
important to set up
your electrodes
correctly when
running a gel
Red is positive
Black is negative
Predicted
restriction sites
3000 bp
2500 bp
500 bp
http://www.wiley.com/legacy/college/boyer/0470003790/animations/agarose/agarose.htm
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html
Plasmid X
3000 bp