Restriction enzymes bound to then eventually cutted the DNA at a specific nucleotide sequence by cleaving the phosphodiester bonds1. Restriction enzymes bound to specific nucleotide because it is known as sequence specific or as Type II endonucleases1. These enzymes are extracted from numerous bacterial strains in order to determine what bacteria or organism was affecting the DNA sequence. The cleavage of DNA with restriction enzymes lab was conducted to observe the recognition site and cleavage pattern of the organism: Bacillus amyloliquefaciens H (Bam HI) and Escherichia coli RY13 (Eco RI) that affected three different DNA’s. After the procedure has been conducted, the fragments would be examined to observe the base pairs of the DNA. Most of the recognition sites shown partial cleavage or complete cleavage.
Restriction enzymes cut the DNA at a specific nucleotide sequence by binding to the site first then cleaved the phosphodiester bonds1 after cutting the DNA. Therefore, double stranded recognition …show more content…
Then five reaction tubes were used to mix reaction buffer, qualified water, Bam HI or Eco RI, and DNA 1, DNA 2, or DNA 3 to reach a final volume of 30 µl. The restriction enzymes were added last to avoid possible errors or inactivation. The each solution were mixed in order to avoid dense layers of enzyme solution at the bottom of the tube. Each tubes were incubated for 60 minutes in a 37oC water bath to guarantee cleavage of DNA. Eventually, 5 µl of 10x gel loading solution was added to each reaction in order to stop the reactions. Then each solutions was mixed again then soon placed in heated bath for two minutes, eventually added to the electrophoresis gel for about 60 minutes. The .8% agarose gel used for electrophoresis aided in slowing the migration of the DNA fragments since large DNA fragments migrate faster than expected because of the strength of the voltage
Restriction enzymes cut the DNA at a specific nucleotide sequence by binding to the site first then cleaved the phosphodiester bonds1 after cutting the DNA. Therefore, double stranded recognition …show more content…
Then five reaction tubes were used to mix reaction buffer, qualified water, Bam HI or Eco RI, and DNA 1, DNA 2, or DNA 3 to reach a final volume of 30 µl. The restriction enzymes were added last to avoid possible errors or inactivation. The each solution were mixed in order to avoid dense layers of enzyme solution at the bottom of the tube. Each tubes were incubated for 60 minutes in a 37oC water bath to guarantee cleavage of DNA. Eventually, 5 µl of 10x gel loading solution was added to each reaction in order to stop the reactions. Then each solutions was mixed again then soon placed in heated bath for two minutes, eventually added to the electrophoresis gel for about 60 minutes. The .8% agarose gel used for electrophoresis aided in slowing the migration of the DNA fragments since large DNA fragments migrate faster than expected because of the strength of the voltage