Dna Digestion and Electrophoresis

Restriction enzymes are a tool that allows us to pinpoint human identity down to single differences in our DNA. Work through the following simulation so you can see these molecular scissors in action.

Introduction: All organisms have DNA (Deoxyribonucleic Acid) in the nuclei of their cells. The aim of this experiment is to extract this DNA using common household detergent to breakdown the cellular membrane then through the use of ethanol to extract the DNA from the Kiwi and Strawberry fruit. After the DNA has been extracted the DNA will be visualised by agarose gel electrophoresis which is done by adding a dye to the extracted DNA then being placed in the gel, once the negatively charged DNA has been placed in the gel it will slowly move towards the positively charged end (anode) then is viewed under an ultraviolet light then compared to the control of known lengths of DNA to show the size of the unknown DNA samples.

The goal of the restriction digests is to be able to cut the plasmids at specific sites. This step

We placed the gel into the running chamber, and then we completely covered the gel with TAE. 3 microliters of loading dye was added to each tube; this would help distinguish the enzyme from the gel. As before, we tapped the tube on the table to mix. Then we carefully added each of the four samples into their own wells. A total of 33 microliters of each sample was poured into each well. Afterwards, we attached the positive and negative electrodes to their corresponding terminals on the power supply and gel box. We turned on the power to around 80 volts and waited 45-60 minutes for the loading dye to move down the gel approximately 6-8 cm. Finally, we were able to visualize the DNA in the gel and write down the

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

Using the electric current, scientists pass the DNA through gel, and as smaller molecules get through gel quicker than those of bigger sizes, DNA molecules get separated according the sizes of the molecules. We utilize the property that large molecules move slower, and DNA is slightly negatively charged(due to phosphate groups), so it will move to the positive pole of the gel.

· Explain the process of electrophoresis as it relates to separating and identifying different DNA fragments

Gel electrophoresis is a procedure used in laboratories to separate DNA, as well as RNA and proteins. A gel slab is placed in a buffer-filled box and an electrical field is applied. The negatively charged DNA will migrate towards the positively charged side, where it can then be recorded and further analyzed.

Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA

During the course of the next three practical classes you shall be performing a number of techniques in order to isolate and manipulate DNA from bacteria. The practicals are spread over three sessions, the techniques that you will perform are

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of DNA or proteins in a matrix ofagarose. The proteins may be separated by charge and/or size

Gel-Filtration Chromatography is a commonly used method used in order purify a protein from a mixture, by means of separations. Different biomolecules differ in size, or their molecular weight. In the gel matrix inside the chromatography column, there are gel beads which are porous to allow certain sized molecules to enter. The molecules that are able to enter the pores of the gel, are held in stationary phase and will elute from the column later on, these are usually smaller, to medium sized molecules. Larger molecules that are not able to fit in the pores will elute out of the column first, they are involved in mobile phase where they just go straight through the column without interacting with the gel beads. Smaller molecules will have a higher elution volume, while the larger molecules will have a lower elution volume. The volume to elute the protein is inversely proportional to the molecules size.

Electrophoresis is the method of separation of charged molecules based on different migration in an electric field. Applicable features of the electrophoresis technique such as high effectiveness, high resolving power, high speed, fully automation and a variety of injection based pre-concentration schemes and detection modes have all been broadly examined. (Tavares, et al., 2003). It is a developed systematic and micro preparative instrument. The method provides faster separations, at higher resolution and with greater separation efficiencies (Swerdlow & Gesteland, 1990). Capillary electrophoresis has applications in several areas like clinical/forensic, cosmetological, environmental, nutritional and pharmaceutical. (Tavares, et al., 2003). For forensic applications it has exceptional separating power, rapid analysis times, and high mass sensitivity, in terms of reagents and consumables, requiring only least quantities of sample (Tagliaro, Pascali, & Lewis, 2013)

The basic principle of electrophoresis involves the separation and isolation of charged molecules due to their differential migration in a buffer

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double