Molecular Genetics Lab
Review
Bacterial Transformation• Genetic transformation—host organism takes
in and expresses foreign DNA• Genetic engineering—manipulation of an
organism’s genome using DNA technology– Introduction of foreign DNA into organism
• You used antibiotic-resistance plasmids to transform E. coli– If bacteria incorporates the foreign
DNA, they became ampicillin resistant
E. coli
• Most common bacterium in human gut• Studied extensively• Important research organism– Reproduce rapidly– A single cell can divide to form millions of cells
overnight– No nuclear envelope surrounding chromosome– One single chromosome– Some contain plasmids resistant to certain drugs
Plasmids
• Circular pieces of DNA• Outside the main bacterial chromosome• Carry their own genes for specialized functions• Plasmids can be used in genetic engineering to
introduce foreign genes into a bacterium
The Experiment
Results
Gel Electrophoresis
Restriction Enzymes
• Bacteria have enzymes that cut (or digest) the DNA of foreign organisms to protect themselves
• Scientists have isolated several hundred of these enzymes, restriction enzymes
• Each is able to recognize and cut at a specific DNA sequence, the recognition sequence
• DNA fragments can be analyzed and used
The Experiment
• You used restriction enzymes to cut DNA into fragments
• You used gel electrophoresis to separate the samples of DNA that had been cut
• You compared fragments of unknown size to fragments of a known size to calculate the unknown fragment sizes
Restriction Enzymes
• Restriction enzymes are specific to an exact recognition sequence
• Sticky ends & Blunt ends
Gel Electrophoresis
• Separates molecules of the rate of movement through a gel under electricity
• DNA is negatively charged—will move toward positive pole of the gel
• Different sized fragments move at different rates– Smallest move the most quickly, thus migrate the
farthest
Analysis of Results
• Each fragment is a particular number of nucleotides long
• To find out the size of the unknown DNA fragments, you ran it along side DNA with known fragment sizes
• HindIII cut DNA into known fragment sizes• It will determine fragment sizes of DNA cut by
EcoRI
Graphing HindIII