Biochemistry Report
Restriction Endonuclease Digestion of DNA from E. coli cells and Analysis by Agarose Gel Electrophoresis
Introduction
The main goals of this experiment are testing an alternative procedure called “boiling lysis”, evaluating the quality of the purified plasmid for restriction digests, and identifying the mislabeled plasmid.
The plasmid DNA from a carrier E. coli strain was purified by the boiling lysis. In the boiling lysis method, the bacterial cells were given momentary heat treatment in boiling water in presence of lysozyme and triton X-100. Since the plasmid DNA is small in size, it comes out from the bacterial cell, while the genomic DNA remains trapped in the cell. Successive high-speed centrifugation separated the plasmid DNA from rest of the cell debris, which form pellet. The pellet was removed and plasmid DNA was recovered by isopropanol precipitation method.
Restriction enzymes are enzymes that selectively cut a DNA molecule at a particular place called restriction sites. Restriction enzymes also recognize a specific sequence of nucleotides, which vary between 4 and 8 nucleotides, in particular, palindromic. These restriction enzymes were kept in a 50% glycerol buffer that does not freeze at -20 oC. The four available enzymes used for this experiment were EcoRI, AvaI, HincII, and RsaI. The lengths of the DNA fragments from the restriction digests are used to map the relative locations of the four available enzymes in the plasmid. From this restriction sites, the unknown plasmid will be identified.
Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. The length of the fragments from digestion is estimated by comparing the results obtained from the reference DNA fragments.
Materials and Methods
Plasmid purification. The bacterial culture used for this experiment is JM109 strain of E. coli carrying an unknown plasmid. For 20 seconds, 1.5 mL of bacterial culture was
Introduction
The main goals of this experiment are testing an alternative procedure called “boiling lysis”, evaluating the quality of the purified plasmid for restriction digests, and identifying the mislabeled plasmid.
The plasmid DNA from a carrier E. coli strain was purified by the boiling lysis. In the boiling lysis method, the bacterial cells were given momentary heat treatment in boiling water in presence of lysozyme and triton X-100. Since the plasmid DNA is small in size, it comes out from the bacterial cell, while the genomic DNA remains trapped in the cell. Successive high-speed centrifugation separated the plasmid DNA from rest of the cell debris, which form pellet. The pellet was removed and plasmid DNA was recovered by isopropanol precipitation method.
Restriction enzymes are enzymes that selectively cut a DNA molecule at a particular place called restriction sites. Restriction enzymes also recognize a specific sequence of nucleotides, which vary between 4 and 8 nucleotides, in particular, palindromic. These restriction enzymes were kept in a 50% glycerol buffer that does not freeze at -20 oC. The four available enzymes used for this experiment were EcoRI, AvaI, HincII, and RsaI. The lengths of the DNA fragments from the restriction digests are used to map the relative locations of the four available enzymes in the plasmid. From this restriction sites, the unknown plasmid will be identified.
Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. The length of the fragments from digestion is estimated by comparing the results obtained from the reference DNA fragments.
Materials and Methods
Plasmid purification. The bacterial culture used for this experiment is JM109 strain of E. coli carrying an unknown plasmid. For 20 seconds, 1.5 mL of bacterial culture was