Ava 2 Lab Report

2. Place a large“+” on the end of the gel diagram where the positive electrode would go. Place a large

In the experiment we used Turnip, Hydrogen Peroxide, Distilled Water, and Guaiacol as my substances. On the first activity, Effect of Enzyme concentration of Reaction Rate for low enzyme concentration, we tested three concentrations of the turnip extract, and hydrogen peroxide. For the Turnip Extract I used 0.5 ml, 1.0 ml, and 2.0 ml. For hydrogen peroxide we used 0.1 ml, 0.2 ml, and 0.4 ml. We used a control to see the standard, and used a control for each enzyme concentration used. The control contains turnip extract and the color reagent, Guaiacol. We prepared my substrate tubes separately from the enzyme tubes. My substrate tube

The first agarose gel provided insight into whether or not Zassy’s genomic DNA had been cut by the Sal I HF (NEB) restriction enzyme. As seen in Figure 1, Zassy’s cut DNA, in lane 3 left clear fragments between the 3 kb and 0.5 kb areas. Zassy’s uncut DNA, in lane 2, left a clear and bright band above the 10 kb marker. Therefore, the gel confirmed that Zassy’s DNA was successfully cut with Sal I because there was a significant difference the cut and uncut DNA that was run through the gel. There was an additional lane of cut DNA because another student adopted Zassy in the early stages of this experiment. Nevertheless, the clear distinction between lanes 2 and 3 allowed the experiment to continue on to purification of the DNA.

At the beginning of the ADI silica lab, we were asked the question “Which “magma” leads to more explosive eruptions and is potentially more dangerous to humans?” . We would soon experiment with “silica” and liquid to determine how the silica content affects its viscosity and relates to potential dangers of a volcanic eruptions. For this experiment to be successful, you must know some key background information. Silica is a solid compound which is present in magma and influences its properties, viscosity is what we call a fluid's resistance to flow. These two core idea’s work together; high silica equals high viscosity, which traps more gases, and leads to a more explosive volcanic eruption. On the other hand,

The purpose of the DNA restriction and electrophoresis lab was to first become familiar with the properties of restriction enzymes and discover that they, along with agarose gel electrophoresis, are used to characterize DNA molecules. Restriction enzymes are used to make cuts or join together DNA fragments. The cuts result in either staggered cleavage, or blunt cleavage. Staggered cleavage results when the breaks are offset. This cleavage usually results in sticky end being produced because for each DNA end that is produced there are extensions with unpaired nucleotide bases. Blunt cleavage results when the breaks in the phosphodiester bonds are right across from each other.

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

Restriction endonucleases are proteins that cleave DNA at specific restriction sites; the protein recognizes specific base pairs on the DNA and cuts

30uL of the LB Buffer added to 30uL of each saved samples). Similar addition of LB Buffer (10uL) was made with the Protein Standards Ladder (10uL). After the addition of the LB Buffer to the appropriate samples, it was placed on a 95oC hot plate for 8 minutes (note: it was instructed to be for 5 minute). This was followed by the preparation of the gel setup (i.e. removing the tape at the bottom of the precast gel and adding the gels at the back and front of the apparatus gel). To this setup, 50uL of 10X SDS running buffer + 450uL of deionized water (making it a 1X Buffer) to the outer chamber was added. In the inner chamber, 150uL of 1X SDS (1X was made from the 10X SDS buffer, the same way by diluting it with a 1:9 ratio of deionized water) running buffer was added. With such setup and the 5 samples that was heated in 95oC, the lanes—starting with lane 1—were pipetted in the following order: 10uL of Protein Standards sample (lane 1), 20uL of “+IPTG with Elution Buffer” (lane 2 and 3), 20uL of “-IPTG with Elution Buffer” (lane 4 and 5), 20uL of “+IPTG Supernatant” (lane 6 and 7), and 20uL of “-IPTG Supernatant” (lane 8 and 9). After loading the gel in such manner, the start of running the gel was done at 200V (making note of it being stable initially), and it was stopped till the bromophenol blue bands reached ~1cm

After the completion of electrophoresis, the gel is placed in denaturing buffer for half an hour and then placed into neutralizing buffer. After the gel is neutralized, it is placed onto a sponge wick sitting in salt solution. A nylon membrane is then placed over the gel. A stack of paper towels is then placed over the membrane. Sometimes a layer of glass is placed over the towels and weights are stacked onto the glass. This would ensure that the gel and membrane has even contact, but to much weight would result in crushing the gel. Once this is set up, the transfer is left overnight. During this time, capillary action with pull the salt solution in the dish below up through the gel. This process transfers DNA fragments from the gel onto the membrane filter. Any weights and the paper towels are removed the next morning. The nylon membrane is then removed and then rinsed with neutralizing buffer. The membrane is then placed on a UV transilluminator for cross linking. Once the membrane is removed from the transilluminator, the DNA is ready for hybridization.

1. Restriction enzymes cut DNA molecules at specific locations. They search for a particular nucleotide sequence, then stop and slice the strands.

Lab 4A consists of using gel electrophoresis to further examine the pARA-R plasmid by viewing them in a DNA ladder. Start by adding loading dye into the two tubes and centrifuging them for a few seconds. Before putting them in the gel walls check to make sure it is in the negative side and not the positive side. Add buffer to cover the walls and keep track of where you will add the R-, R+, and ladder. It is very important to use new, fresh tips and to add them very carefully. Once everything has been put in, cover the box and connect the cables to the power supply in the correct order: black to black for negative and red to red for positive. Turn the power on and put the voltage up to 130v-135v and let it set for 40 to 50 minutes. For the first two to three minutes check to see if it is moving towards the positive side and if it isn’t, inform the instructor. This lab ends with the instructor providing a picture of how far the plasmids went.

Restriction enzymes (or restriction endonucleases), originally isolated from Haemophilus influenzae in 1970, are enzymes within a cell that cleave foreign DNA within a specific and predictable nucleotide sequence (known as a restriction site) regardless of the source of such DNA. Such restriction sites generally are four to eight base pairs in length. It is thought that, together with enzymes that methylate portions of native DNA, restriction enzymes protect cells from DNA of invading organisms cutting such DNA into pieces, thereby

A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides known as recognition sites.

Then solution III is added which contains 3.0 M potassium acetate at pH 5.2. This solution causes the pH to reduce and aids with the reassociation of the plasmid DNA as they are inter wound covalently. In this section a separation occurs where the chromosomal DNA that 's clumped is found at the bottom of the test tube. While the plasmid remains in the solution and the chromosomal DNA is at the bottom of the test tube. Then the test tube is centrifuged

In Figure 1, the photograph shows how each of our samples ran in the agarose gel. We can see different bands in different wells, meaning that the enzymes were working and that the plasmid has been cut in different patters according to the reaction. Before starting measuring the distances and doing the calculations, it is noticeable that the bands of the ladder were not very well separated. Therefore, it will not be accurate to rely on those bands to make the standard curve and determine the sample fragment sizes. The unclear ladder band could be a result of different factors, including the amount of time the gel was running for, the components of the buffer, the gel concentration or even the voltage and current conducted. Therefore, we can say that the digestion had worked, since we are able to see different fragments in the samples, but the electrophoresis was not very successful as the ladder bands were not separated very well, in addition to their general blurriness.