Dna Extraction Lab Report

1. If each individual has such a small amount of DNA, how do the bands on the gel contain enough DNA to be visible?

In this experiment, DNA was extracted from human cheek cells using Gatorade, soap solution, and rubbing alcohol. To do this, Gatorade was swished in the subject’s mouth vigorously for 45 seconds, and then added to soap solution and rubbing alcohol. This mixture was left to sit in ice water for two to three minutes. It was hypothesized that when cheek cells were placed into Gatorade, soap solution, and rubbing alcohol, that the DNA would be extracted, forming a stringy substance. This hypothesis was supported by the results of the experiment. After adding the alcohol to the Gatorade and soap solution, a semi-translucent, fibrous substance was formed.

12.Stir then pour 2.5ml of the enzyme mixture into one of the test tubes not allowing any water from the saucepan into the test tube.

There are three specific steps required to isolate DNA from its cellular contents. The steps used to remove and expose DNA from its cell are: breaking down the food type you are using by crushing it, for example a banana or strawberries, exposing the substance to a sodium chloride (NaCl) solution, subjecting the product to detergent solution (dH2O), filtering the solution and lastly, the addition of ethanol. When beginning with a solid substance, such as a banana, crushing the substance allows for

To ensure the experiment was kept as a fair test a number of variables were controlled. The temperature of the solutions was kept constant by placing the boiling tubes into a test tube rack and setting it into a water bath with a fixed temperature of 25oC. The temperature needed to be kept low and fixed as a high temperature would denature the enzymes, they would therefore be unable to break down the gelatine and no results would be produced from the experiment. Keeping a constant temperature also meant that the solutions reacted at the same rate.

We did not modify the experiment because it worked perfectly. As soon as we poured the alcohol into the pea mixture test tubes the DNA began forming and becoming visible. Due to this, we did not find it necessary to modify 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.

Moreover, as more cells are broken down the more DNA will be isolated. Then, mixed the KAc/LiCl working solution thoroughly and waited for 10 minutes. Separation of cell debris from the nucleic acid occurred during this part of the procedure. Added EtOH so that the pellet will move through the EtOH and recovered the pellet.

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.

To begin the experiment the restriction enzyme digestions for the suspects had to be made. So, the team gather four micro test tubes and labelled them from 1-4 with one of the team members name on all the tubes. Using a micropipette 10-µl of the enzyme reaction buffer was placed in each of the tubes. One of the team members made sure to tap each of the tubes against workbench to be sure that the buffer all collected to the bottom. Once that was completed, in micro test tube 1 and 2 15-µl of DNA 1 was dispensed, while in test tubes 3 and 4 15-µl of DNA 2 was dispensed.

The reason for that is that I believe the TBE buffer in the 1.5% agarose was a little strong which caused the solution mixed into the agarose to spread all over which caused the UV lights to not pick up the concentration of the DNA. As I need more research I came up on the reasoning as to why we use agarose gel electrophoresis, it is because it is more effective with the separation of DNA fragments. That explains the reasoning as to why the separation did not work as well with the 1.5% agarose gel it may have been either a little strong or weak. The gel that came out better was the 3% agarose gel because of the strong concentration of the gel. With troubleshooting agarose gel the important things to know is that if the gel is faint or no bands then there was not enough concentration of DNA and you want to add more DNA.

The gel was covered with a buffer and then six samples labeled A-F were deposited into the wells using a micropipette. Three of these samples (A, B, C) were control samples to compare to D, E, and F (the mother, child, and father). The safety cover was placed on the unit and then brought to a power source. The leads were connected the chamber and left for approximately 20 minutes. The agarose gel was removed from the tray and placed onto a sheet of plastic wrap. An Ethidium Bromide card was placed face down onto the gel to stain for approximately 5 minutes. Finally, the card was removed and the gel was placed on top of a UV light. The samples were pushed towards the center due to opposite electric charges. Agarose gel separates the DNA samples by the way they were cut. The restriction enzyme MST II cuts the DNA strand at CC/TNAGG where N is any nucleotide base. If the enzyme recognizes this, it is cut. If it does not recognize it then the strand is left whole. We were able to observe the DNA strands due to them being dyed and placed over a UV light. The control samples were utilized so that the other samples could be compared to test for their genotype. The data was analyzed in this way to differentiate between the different genotypes and the number of bars they

2. Measure 5mL of a solvent (water) and pour into different test tubes 6 times using a measuring cylinder.

Plasmids were extracted using the three-step alkaline lysis procedure as outlined by Kado and Liu, 1999. The extracted plasmids were then digested using 1 U of each XhoI and XbaI, respectively according to manufacturer’s (New England Biolabs) recommendation and ran on a 1% Tris Acetate EDTA (TAE) agarose gel. 1 μl of BioLine Hyperladder I was used as the DNA marker.

To find the effect of temperature on the activity of an enzyme, the experiment deals with the steps as follows. First, 3 mL if pH 7 phosphate buffer was used to fill three different test tubes that were labeled 10, 24, and 50. These three test tubes were set in three different temperature settings. The first test tube was placed in an ice-water bath for ten minutes until it reached a temperature of 2° C or less. The second tube’s temperature setting was at room temperature until a temperature of 21°C was reached. The third tube was placed in a beaker of warm-water until the contents of the beaker reached a temperature setting of 60° C. There were four more test tubes that were included in the procedure. Two of the test tubes contained potato juice were one was put in ice and the other was placed in warm-water. The other two test tubes contained catechol. One test tube was put in ice and the other in warm water. After