Unformatted text preview:

3.034 FALL 2005 ORGANIC AND BIOMATERIALS CHEMISTRY LABORATORY EXPERIMENT 3 Engineering DNA via Restriction Enzymes PreLab Questions: 1. What is the definition of an enzyme? Describe the function of restriction enzymes? 2. What is a plasmid? WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high levelof safety training, special facilities and equipment, and supervision by appropriate individuals. You bear the soleresponsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have noresponsibility, liability, or risk for the content or implementation of any material presented. Legal NoticeI. Introduction Enzymes are proteins that serve a specific function: They catalyze chemical reactions, meaning that they initiate / facilitate reactions without being consumed or altered significantly in the process. Enzymes are typically named by the following convention: Substrate + ase. For example, an enzyme that catalyzes degradation of another protein is called a proteinase or protease. An enzyme that digests milk sugar (lactose) is called lactase. In this laboratory, you will learn how to visualize the effects of an enzyme that ligates (cuts) DNA into small fragments through gel electrophoresis. Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides, or DNA bases A, T, G and C. For example, E. coli bacteria that carry the HindIII gene from the bacterium Haemophilus influenzae Rd can be used to produce an enzyme named HindIII (“HIND THREE”) that cuts double-stranded DNA into double-stranded DNA fragments wherever it encounters the sequence: The cut is made between the adjacent A and T, as shown by the arrows. This sequence is found in many DNA, including linear, double-stranded DNA isolated from the bacteriophage lambda (Lambda DNA) which has 48,502 base pairs. Treatment of Lambda DNA with this enzyme produces exactly 7 fragments, each with a precise length and nucleotide sequence. This fragmentation of the DNA via the restriction enzymes is called restriction enzyme digestion, and occurs during incubation of the enzyme with the double-stranded DNA molecules in a NaCl buffer of concentration and temperature optimized for the enzyme activity. These fragments can be separated from one another through gel electrophoresis, a technique that separates charged molecules under the influence of an electric field. The sequence of each fragment is determined by the molecular weight of each fragment and a priori knowledge of the cutting site of the particular restriction enzyme used. You will demonstrate the effect of this restriction enzyme digestion for a plasmid (circular rings of double-stranded DNA) called pBPZ, analyzed via gel electrophoresis. Since the lengths of the Lambda/HindIII digest are well known, you will use the fragment lengths (“bands”) from the Lambda digest as a marker lane or “ladder” , to determine the segment lengths of the pBPZ digest. In gel electrophoresis , the gel is made from agarose dissolved in a gel stain solution [SYBR safe DNA gel stain]. The gel stain binds to nucleic acids and fluoresces under UV illumination, facilitating the visualization of the DNA fragments. 3.034 FALL 2005 2 LABORATORY 3II. Objectives The goal of this laboratory experiment is to: • Analyze through agarose gel electrophoresis the effects of a DNA restriction enzyme on the size of two types of virus DNA (λ-phage and pBPZ). • Construct a new plasmid to produce the protein Green Fluorescent Protein (GFP). 3.034 FALL 2005 3 LABORATORY


View Full Document

MIT 3 034 - Study Guide

Documents in this Course
Load more
Download Study Guide
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Study Guide and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Study Guide 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?