Lab 6 - Lab 6 Cloning DNA Electrophoresis Complementation...

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Lab 6 Cloning DNA Electrophoresis Complementation Score growth on SD plates Vibrio isolation Re-streak on SWC plates Fly genetics Answer keys for pre-labs and worksheets are posted in the hallway! Come to office hours in VLSB 2084!
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DNA Electrophoresis Load the gel: Ethidium Bromide is a mutagen! Wear GLOVES!!! Add 1 μ l loading dye to your digestion reactions (contains glycerol and bromophenol blue, ~ 300bp) Start loading the gel using the diluted dye (for parcticing) Load 11 μ l of your digestion reations into each well Check polarity (negative charged DNA migrates to Anode (+) Set power supply to 30 V
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R L SC Separation depends on: Polarity (DNA negatively charged) DNA conformation (linear vs relaxed vs super-coiled) Size (big fragment migrate slow) Matrix (% agarose, acrylamide) Standard curve: use fragments of known size
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Marker: make a standard curve (distances from well to each band) Your plasmid (size =distance migrated use standard curve) + control
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DNA electrophoresis, gels + restriction map, DNA conformation DNA: circular (relaxed and super coiled) vs linear. EtBr fluorescence is proportional to the number of nucleotides (bigger fragments = more nucleotides = higher fluorescence intensity OR more DNA = more nucleotides = higher fluorescence intensity), loading dye contains bromophenol blue (negative charge, migrates ~ 300 bp) and glycerol (increases density of DNA so that the DNA sinks into wells of gel). http://en.wikipedia.org/wiki/Gel_electrophoresis Restriction enzymes normally generate “sticky” ends (those overhangs that anneal = hydrogen bonds). Some enzymes also generate “blunt” ends (without overhangs = no hydrogen bonds). The enzyme ligase can form phosphodiester bonds between blunt ends (http://en.wikipedia.org/wiki/Blunt_end)
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Complementation Score for growth downstream of the mating point.
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This note was uploaded on 10/29/2009 for the course BIO biology taught by Professor Meighan during the Fall '09 term at University of California, Berkeley.

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Lab 6 - Lab 6 Cloning DNA Electrophoresis Complementation...

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