Fruit Fly Lab Report

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

We were using a restrictive enzyme to cut the DNA into smaller fragments. For the restriction digest we pipet 4 micrometers of enzyme mix into the bottom of each of our colored tubes making sure to use a new tip for each sample. Next, we capped the tubes and mixed the contents by flicking the tube a little bit with our fingers. After we mixed the contents, we tapped the tube to make sure all the liquid would go to the bottom of the tube. Then, we put the colored tubes in the heating blocks and we let them incubate for 35 minutes at 37 degrees Celsius.

nant research, to achieve recombinant DNA. The gene that is the focus of this experiment and is

Introduction For centuries, researchers have used Drosophila melanogaster, the common fruit fly, to study genetics. The benefits of using the fruit fly includes: its relatively short generation time, its large amount of available offspring for data, it is easy to store and handle in the laboratory and it is easily and cheaply obtained. Cross-breeding of four types of fruit flies were used in this experiment including: wild type males with normal wings vs. vestigial wing females, wild type males with red eyes vs. white eyed females, wild type male with red eyes vs. sepia eyed females, and wild type males vs. wild type females. In basic mendelian genetics, the terms dominant, recessive and sex-linked are used to describe the different types

Introduction: Drosophila is the name given to a genus of small flies within the family, Drosophilidae. They are commonly referred to as fruit flies or pomace flies. They have historically been called vinegar or wine flies due to the fact that they commonly gather around rotting fruit. The fruit fly is sometimes mistaken for the Tephritidae family of flies which is closely related and is even sometimes referred to as the true fruit fly however; tephritids tend to only feed on unripe fruit, and have been regarded as destructive agricultural pests. For instance, the Mediterranean fruit fly has devastated the agricultural economy sector of several Mediterranean countries millions of dollars in ruined crops.

It takes approximately 10 to 14 days for the fruit fly to go from an egg to an adult fly. Since the life span of the D. melanogaster is very short, we are able to observe multiple generations in a short amount of time. With the fruit fly, we would be able to observe a mutation over several generations in order to get a better understanding of how it affects each generation. Each step within the development of the D. melanogaster is easily observed under a microscope. This allows us to observe if a mutation has an effect on any of the stages during development. These flies are able to breed and reproduce over a short time span. Female D. melanogaster are able to hold the sperm of multiple males and lay over hundreds of eggs, making it very simple to collect and observe many progeny. The D. melanogaster are also ideal organisms because the cultures used to cultivate the flies are small and easy to store and cost efficient. Each vial, if taken care of properly are viable for

Abstract Drosophila melanogaster have been tested on by scientists for over a hundred years. Research on these flies have come to be the foundation of genetic studies everywhere (“Drosophila melanogaster”). In this experiment two environments were created where wild type and vestigial Drosophila melanogaster reproduced over multiple generations. One environment has a simulated predator and the other does not.

No vial was put upright since sleeping flies would fall on the food and die thus the vials were put on their sides. The vials were store in the light and at a temperature of 25 Celsius to 25 Celsius for a week since warmer temperature shorted the generation time and colder temperature resulted in top a longer generation time. The temperature was recorded and in the Result

fischeri genomic DNA and pUC 18 vector were ligated with T4 DNA ligase. The DNA samples were first placed in 65ºC water bath for 20 minutes to inactivate the restriction enzyme remained in the samples. Different molar ratios of insert-to-vector were prepared for this experiment, which includes: 1:1, 2:1, 3:1, and 4:1. An additional 3:1 ratio of lux operon insert to vector was prepared for ligation as well. The mixture was made to a total of 30 µL containing 0.1 µg of vector, 0.3 µg per molar insert, and 6 µL of 5X Buffer.

In this experiment the independent variables were the amount of flies that emerged and the phenotypes of those flies. The hypothesis to be tested was the expected phenotypic ratio of the F2 generation of the unknown true bred cross would be 9:3:3:1. To complete this experiment a culture vial must be made for the flies to inhabit. The flies were then incubated for two weeks to go through a complete life cycle. After two weeks, the flies were obtained and observed under a light microscope to conclude phenotype and be counted.

Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB broth.

The Lambda kit was used to rid the digested pGEM of the stuffer fragment so as to lower the probability of it re-ligating back into the plasmid instead of the luxAB inserts [1]. In total, four ligation reactions were prepared under varying conditions – L1 contained a 1:1 insert to plasmid (with stuffer) ratio, L2 contained a 2:1 insert to plasmid (with stuffer) ratio, L3 contained a 1:1 insert to plasmid (no stuffer) ratio, and L4 contained a 2:1 insert to plasmid (no stuffer) ratio. Strains of highly competent Zippy cells, derived from E. coli DH5 cells, were diluted and transformed with the recombinant plasmids [1]. pGEM contained the Ampr gene which inhibited ampicillin via -lactamase, thus, DH5 cells that took up pGEM were able to actually grow in the agar plates containing ampicillin [1]. In order to determine the efficiency of the ligations and observe any activity of luciferase, several transformations were plated, and the produced blue and white colonies were counted [1]. If pGEM contains an insert, the insert itself will inactivate LacZ by disrupting its sequence -- blue products result from intact pGEM because -galactosidase would properly cleave X-gal, and white products result from cut pGEM because -galactosidase would be non-functional [1]. The negative control was comprised of an aliquot of DH5 cells and sterile water, the positive control was

three specific databases. These organisms have been used as model organisms through multiple clinical researches to inform us about biology. They are ideal organisms due to characteristics such as how roundworms and fruit flies grow to adulthood quickly. In addition, they share principles such as the cell cycle in yeast that is regulated by homologous proteins that are similar in humans; hence they will be able to enlighten us on the genetic profiling in humans. The five most used organisms are yeast, fruit fly, bacteria, mouse, and roundworm. Out of those five most common organisms we used roundworm, yeast, and fruit fly to conduct our analysis. To analyze the organisms we had to use databases specifically for each organism (i.e. wormbase.org, flybase.org, yeastgenome.org). We also used the ApE software to analyze gene sequences whose information was not readying available in the databases. From these databases we were given the information needed to analyze each organism.

Much can be learned from studying an organisms DNA. The first step to doing this is extracting DNA from cells. In this experiment, you will isolate DNA from the cells of fruit. Materials (1) 10 mL Graduated Cylinder(2) 100 mL Beakers15 cm Cheesecloth1 Resealable Bag1 Rubber Band (Large. Contains latex pleasewear gloves when handling if you have a latex allergy).Standing Test TubeWooden Stir StickFresh, Soft Fruit (e.g., Grapes, Strawberries, Banana, etc.) ScissorsDNA Extraction SolutionIce Cold EthanolYou Must ProvideContains sodium chloride, detergent and waterFor ice cold ethanol, store in the freezer 60 minutes before use. Procedure If you have not done so, prepare the ethanol by placing it in a freezer for approximately 60 minutes.

The chemical and reagents used for the extraction and quantitation of DNA were: Plant DNAzol (0.3ml/0.1g), 100% ethanol (100%: 0.225 ml/0.1 g, 75%: 0.3 ml/0.1 g), Chloroform (0.3 ml/0.1 g), Plant DNAzol-ethanol solution: Plant DNAzol, 100% ethanol (1:0.75 v/v), TE buffer (10 mM Tris, 1 mM EDTA pH 8.0), 1.2% agarose gel (Agarose, 1X TAE buffer), 6X loading buffer (glycerol, Tris/EDTA pH 8.0, ethidium bromide), .25X TAE buffer, Restriction enzymes and Restriction endonuclease buffers. All the chemicals used were quality grade. The restriction