Double Digest Lab

1. From what organism are restriction enzymes derived? What role do these enzymes play in this natural host?Phages. Enzymes

First, 50 uL of bacterial cells were made competent by being treated with 590 uL of CaCl2 in three different test tubes before transformation of the plasmids took place. In order to enhance the uptake of the plasmid DNA the instructor added the CaCl2 solution to the bacterial cells. The tubes were placed into a cold ice bath for about ten minutes after the CaCl2 solution was added into the test tubes of E. Coli.

Digestion is a complicated process that uses many different processes to digest food efficiently. It is necessary for not only us but for almost every organism. A major part in digestion is pH or how acidic or basic a substance is. pH helps digestion happen, the question is for the Stentor and the Rotifers at what pH ranges does their digestion occur? We will test that by using pH indicators, and observing the digestion happen under the microscope.

For this experiment, we used two restriction enzymes called Ava II and Pvu II. Four tubes were needed for the experiment as well. Each microtubule was labeled accordingly, A, B, and C. Using Micropipettes, we added 2 microliters of pUC19 DNA into each tube. To make sure the DNA was on the bottom of the tube, we tapped each tube on the lab bench. Each tube had its own specific amount of different solutions added on, however the final volume for each tube was 30 microliters. Tube A had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Ava II enzyme. Tube B had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Pvu II enzyme. Tube C had, 23 microliters of DI Water, 3 microliters of the buffer, 1 microliter of Ava II, and 1 microliter of Pvu II. The last tube C had, 25 microliters of DI water, 3 microliter of the buffer, and no enzymes. Each solution was added according to how it is written here. After we finished adding the solutions to each tube, we tapped the tubes on the lab bench to make sure the

The pH was tested by adding acid (HCl), base () and neutral (water) to the enzyme. When the enzyme samples were tested using the hydrogen peroxide and O2 sensor test it was determined that any

In part II of the lab six small glass tubes were obtained in a test tube rack. Ten drops of distilled water were then added to test tube 1, five drops to tubes 2-4, and no drops in tubes 5 and 6. Five drops of 0.1M HCl were added to test tube 5 and five drops of 0.1M NaOH to test tube 6. Five drops of enzyme were then added to all tubes except tube 1. Tube 3 was then placed in the ice bucket and tube 4 was placed in the hot bucket at 80-900C for five minutes, the remaining tubes were left in the test tube rack. After the five minutes five drops of 1% starch was added to every tube and left to sit for ten minutes. After ten minutes five drops of DNSA were then added to all the tubes. All the tubes were then taken and placed in the

The enzyme that will be focused on is the Beta-lactamase enzyme. There exist many isozymes including Beta lactamase (Salmonella enterica subsp. enterica serovar Enteritidis str. 2010K-0284) and Beta lactamase (Thermoanaerobacter sp). The enzyme that will be reported on is the type TEM-1 Beta-lactamase (1).

The purpose of this lab is to discover how changes in temperature can affect enzyme activity. All walks of life, from humans to plants, are living because of biochemical reactions. Biochemical reactions are processes that occur on a cellular level and are responsible for sustaining life: Photosynthesis, Cellular Respiration, Digestion, etc. Within these reactions, molecules change their identity and transform into new molecules. In the middle of these molecular transformations are enzymes (sites.com).

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

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

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

Add 5mL of gastric juices (contains both pepsin solution and HCl) to test tube 3

3. The tubes are allowed to incubate in a 37˚C water bath for 1 hour. The final pH of the solutions is tested and the amount of protein digestion is estimated using a scale of (+++), (++), (+), and (-) by

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