Before you get started...try this and learn about the equipment and process
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WET LAB VERSION:
FOLLOW THE INSTRUCTIONS GIVEN IN CLASS FOR THE PARTICULAR DESIGN OF ELECTROPHORESIS APPARATUS (SEE PHOTO ABOVE, LEFT), THE PARTICULAR MICROPIPETOR MODEL AND PRACTICE DYES AND THE PARTICULAR SAMPLE DNAS (BACTERIOPHAGE LAMBDA DNA, CUT WITH VARIOUS RESTRICTION ENZYMES) THAT ARE AVAILABLE. A GOOD BASIC GEL RECIPE IS A 1% AGAROSE IN A 1X TBE (TRIS-BORATE-EDTA) BUFFER. FOR THE CONCENTRATIONS OF PRECUT DNA AVAILABLE COMMERCIALLY, A GOOD LOADING VOLUME IS 5-10 MICROLITERS.
ONCE YOUR GEL WITH DNA SAMPLES HAS RUN AND BEEN STAINED WITH THE METHYLENE BLUE, MAKE A SKETCH (OR A PHOTO IF A CAMERA IS AVAILABLE) AND COMPARE THE PATTERN OF RESTRICTION FRAGMENTS (RFLP'S - SEE PHOTO ABOVE, RIGHT) THAT YOU SEE WITH WHAT IS EXPECTED FOR THE GIVEN RESTRICTION DIGEST OF LAMBDA PHAGE DNA.
ASSIGNMENT: TURN IN YOUR PHOTO OR SKETCH (USE ANOTHER GROUP'S IF YOUR BANDS WERE TOO FAINT TO SEE) AND ANNOTATE THE IMAGE WITH THE SIZES THAT YOU DEDUCED FOR EACH BAND, BASED ON COMPARISON TO A DIGEST PATTERN THAT YOU FOUND IN A WEB SEARCH FOR THE SAME RESTRICTION ENZYME.
FOLLOW THE INSTRUCTIONS GIVEN IN CLASS FOR THE PARTICULAR DESIGN OF ELECTROPHORESIS APPARATUS (SEE PHOTO ABOVE, LEFT), THE PARTICULAR MICROPIPETOR MODEL AND PRACTICE DYES AND THE PARTICULAR SAMPLE DNAS (BACTERIOPHAGE LAMBDA DNA, CUT WITH VARIOUS RESTRICTION ENZYMES) THAT ARE AVAILABLE. A GOOD BASIC GEL RECIPE IS A 1% AGAROSE IN A 1X TBE (TRIS-BORATE-EDTA) BUFFER. FOR THE CONCENTRATIONS OF PRECUT DNA AVAILABLE COMMERCIALLY, A GOOD LOADING VOLUME IS 5-10 MICROLITERS.
ONCE YOUR GEL WITH DNA SAMPLES HAS RUN AND BEEN STAINED WITH THE METHYLENE BLUE, MAKE A SKETCH (OR A PHOTO IF A CAMERA IS AVAILABLE) AND COMPARE THE PATTERN OF RESTRICTION FRAGMENTS (RFLP'S - SEE PHOTO ABOVE, RIGHT) THAT YOU SEE WITH WHAT IS EXPECTED FOR THE GIVEN RESTRICTION DIGEST OF LAMBDA PHAGE DNA.
ASSIGNMENT: TURN IN YOUR PHOTO OR SKETCH (USE ANOTHER GROUP'S IF YOUR BANDS WERE TOO FAINT TO SEE) AND ANNOTATE THE IMAGE WITH THE SIZES THAT YOU DEDUCED FOR EACH BAND, BASED ON COMPARISON TO A DIGEST PATTERN THAT YOU FOUND IN A WEB SEARCH FOR THE SAME RESTRICTION ENZYME.
Eco RI in left two lanes, Pst I on right two lanes, 1 % agarose/TBE gel, 5-10 microliters DNA
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lambda/EcoRI
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lambda/PstI
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lambda/HindIII
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Dry lab version:
click to link to demonstration exercise
