Restriction enzyme

1. Where do restriction enzymes come from and what is their original purpose? Restriction enzymes come from bacteria. Bacteria used restriction enzymes to cut up DNA into fragments to fight off viruses. 2. a. Why do you get different fragment sizes if you cut the DNA from two different species with the same restriction enzyme? Each species has a different arrangement of bases, and the restriction enzyme only cuts after a specific order of bases. Due to that being the case, the specific order of bases won’t be in the same exact spot for each species, so the DNA will be cut in different places. The DNA being cut in different places result with different fragment sizes. b. Why do different restriction enzymes give you different sets…

Edgar Huichapa DNA Mapping Lab 4-29-15 Analysis of Plasmid DNA by DNA Mapping Introduction Restriction mapping is the process of obtaining structural information on a piece of DNA by the use of restriction enzymes (Restriction). Restriction enzymes, also known as restriction endonucleases, are naturally occurring enzymes used in labs today to cut DNA into smaller, more manageable fragments (Bougher). Restriction enzymes are used to cut plasmids which are small DNA molecules that can replicate…

Introduction: In this lab experimentation, the group conducted Deoxyribonucleic acid isolation and restriction analysis on a plasmid from Escherichia coli cells. Plasmids are small circular DNA that are in the bacterium cells. Escherichia coli is a gram- negative bacterium that is known for variable reaction to antibiotics, and can be genetically manipulated. The gram- negative bacterium, Escherichia coli can be genetically manipulated by extracting a certain plasmid that allows it to resist…

Restriction Mapping of Plasmid DNA Preparing 5 Eppendorf Tubes All tubes contain 1 μL of the plasmid DNA, and 2 μL of 10X buffer #2. Tube 1 is the control so there is no enzyme added only 17 μL DI water. For tube 2 the DNA is mixed with 0.5 μL of BamHI and 16.5 μL of DI water, to see total number of base pairs from the linear DNA. In tube 3 DNA is digested with 2 restriction enzymes, which can give the relative distance between the two enzymes. To be more specific, 0.5 μL of Bam HI and EcoRI…

Background DNA molecules can be too big to analyze through electrophoresis, some they must be cut up into smaller pieces. In order to cleave dsDNA at the specific sites, making sure each fragment contain the desired sequence, restriction enzymes are used. Restriction enzymes can recognize a specific sequence of nucleotide and hydrolyze the bond of the DNA backbone. Hence, the fragments from the same DNA source will also be the same if cleave by the same restriction enzymes. Restriction enzymes…

A. fischeriThe overall purpose of this study was to create a genomic library of Aliivibrio fischeri (A. fischeri) thus aiding in creating a restriction map of the lux operon. It also employs typical molecular techniques important for biologists to understand. In this portion of the lab, the chromosomal DNA (chDNA) will be isolated. Its purity will be measured using spectrophotometric analysis. Lastly, the DNA will be digested and verified via gel electrophoresis. A. fischeri is a gram…

Purpose: To study the expression of the Vibrio fischeri luciferase operon in Escherichia coli (E. coli) by isolating and purifying a plasmid from E. coli, determining its orientation with respect to the plasmid backbone by restriction mapping, and transforming it into an alternate E. coli strain. Methods: Each individual received either E. coli A or B strain of pKN800 plasmid DNA. The plasmid was purified and isolated, then cut with PstI restriction enzyme to create a sample of PstI-cut and…

Restriction enzymes are used to manipulate DNA sequences to create recombinant DNA by first cutting up the foreign DNA in order to protect the bacteria cell against invading DNA from other organisms. The enzyme is very specific when it comes to identifying a specific DNA sequence. When the enzyme identifies the specific DNA sequence it cuts both DNA strands at specific points at the restriction site. Lastly, the DNA ligase joins the DNA from two different sources and produces a recombinant DNA…

extra parts of cells that we did not. Then we places 750µl of the supernatant in a spin column and centrifuged it as the DNA would stick to the membrane and the liquid would go through to the tube. After adding wash buffer and centrifuging it a few times to make sure all the liquid was out that we could possible get out we places the spin column on a new tube. Then we added elution buffer to the column to separate the DNA from the membrane. In order to tell if we were successful in obtaining DNA…

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…