Recombinant Dna Lab Report

The purpose of this DNA lab is to create a recombinant DNA molecule by inserting the red fluorescent protein into pARA. Recombinant DNA molecules are formed by laboratory methods of genetic recombination to bring in genetic material from other sources. The two plasmids that were used in the lab were pKAN-R and pARA. The pARA plasmid carries the ampr gene, which produces the protein beta lactamase. Protein beta lactamase is the enzyme that destroys the antibiotic ampicillin. Beta lactamase allows bacteria to reproduce in the company of ampicillin. pARA also carries a gene for the AraC protein. The AraC protein helps the bacterium make proteins encoded by genes put into the plasmid. Because a gene can be expressed if it’s inserted into a particular

DNA ligase is is a type of enzyme that enables the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. The newly formed plasmids represent the recombinant DNA molecules because the four restriction fragments have been changed in different ways to produce new constructs. BamH I and Hind III are the restriction enzymes being used, and cutting the plasmids at the BamH I and Hind III restriction sites leave sticky ends. Sticky ends are the ends of the DNA double helix where unpaired nucleotides extend beyond the other. The sticky ends on the cut DNA are ligated to any other fragment of DNA with a complementary sticky end. pARA has one BamH I and one Hind III restriction site, so the digest will leave two fragments. The large restriction fragments carry the ampr gene. The ampr gene provides resistance to ampicillin. The smaller fragment doesn’t carry any genes. The pKAN-R plasmid has one BamH I and one Hind III restriction site that line the rpf gene. The digestion of pKAN-R leaves two fragments. Ligation bonds any two BamH I sticky ends together and any two Hind III sticky ends together. The best combination to have for this lab is 4018 bp pARA fragment recombined with 702 bp pKAN-R fragment. The pARA fragment contains the ampr gene while the pKAN-R fragment contains the red fluorescent protein. The combination of the two fragments

DNA fragments will migrate through the agarose gel maze. Agarose gel is a polysaccharide matrix that functions as a sieve. DNA is negatively charged because of the phosphate groups, therefore the DNA will move towards the positive electrode. Small molecules will move through the agarose matrix faster than larger fragments because of their size. Smaller DNA fragments move through the agarose molecules faster than the longer fragments. The digested, undigested, and ligated plasmid samples use electrophoresis to separate the pieces. Two or three bands will appear in the undigested plasmid lanes because the plasmids isolated from the cells exist in three forms. The first form is called supercoiled where it’s a circular piece of tubing that’s twisted. The twisting makes a compact molecule that moves quickly through the gel for its size. An open-circle plasmid is when a plasmid encounters a break in one of the covalent bonds positioned in its sugar-phosphate backbone along one of the two nucleotide strands. Recurring freezing and thawing of the plasmid can cause the break. The tension kept in the supercoiled plasmid is released as the twisted plasmid unwinds. The open-circle plasmid will not move through the agarose gel as easily as the supercoiled plasmid. The open-circle will form will be closer to the well than the supercoiled form even though they’re the same size in the terms of base