Cleavage of Lambda Dna with Ecor1 Endonuclease
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Cleavage of Lambda DNA with EcoR1 Endonuclease
Background:
In this lab, the purpose was to learn about restriction enzymes and how to use gel electrophoresis. Restriction enzymes are enzymes that cut single or double stranded DNA at specific nucleotide sequences, or restriction sites. They are found in many kinds of bacteria, such as blue-green algae. Restriction enzymes are needed for molecular cloning, mapping, and sequencing the genetic structure. Without restriction enzymes, the Human Genome Project would have not been completed. They are named based on the organism on which they live. Scientists use the first letter of the genus, then the first two letters of the species. The form of strain or sub-strain at times follows the species. Finally, a Roman Numeral is added to the end for the purpose of designating the enzymes from one another if they were created by the same organism or by different sub-strains. EcoR1s name comes from Escherichia coli (use of E from genus and co from species), strain RY (use of first letter R), and Roman Numeral 1 (distinguishes it from other restriction enzymes found in Ecoli or in that strain). Restriction enzymes are necessary for this lab because they are needed to cut the DNA at its restriction sites so the DNA can go through the agarose gel. A palindrome is a specific nucleotide sequence that is recognized by the restriction enzyme. A recognition site is where the restriction enzymes cuts the DNA and they are usually 4-8 base pairs long. The recognition site for EcoR1 is GAATTC (5 to 3 direction) and CTTAAG (from 3 to 5 direction). Lambda DNA has five recognition sites for EcoR1, resulting in six DNA fragments. The source of Lambda DNA is from the E. coli bacteriophage lambda. The agarose gel acts like a sieve for the DNA fragments. It is a powerful tool of separation, often used for the examination of DNA fragments, which came from the restriction enzymes. The gel pulls the DNA fragments through microscopic pores, letting smaller fragments pass and larger fragments staying towards the front of the gel, a lot like a sieve. As the percent concentration of agarose increases, the smaller the separation is between DNA fragments in the gel. We use .8% agarose because the distance between the DNA fragments was not too large or too small. DNA has a strong negative charge when the pH is neutral. The current running through the gel allows the DNA fragments to separate because the negative charge of the DNA is attracted to the end of the gel, where the positive electrode is located. The smaller the fragment of DNA, the faster/farther the fragment travels through the gel. If there are fragments that are the same size, they move together through the gel. This is known as a doublet.
Hypothesis:
If the restriction enzymes are present, then the