The Methods Of The Electrophoresis

1. List the distances traveled (in mm) for the bands in the DNA Ladder in the table below.

Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.

The proteins are also added to a Laemmli sample buffer in order to give each protein a negative charge so it is able to get pulled through the polyacrylamide gel. The next step is to put the gel into the electrophoresis module and to run it. It is run until the proteins have almost reached the bottom of the gel. A blue tracking dye is added to the Laemmli sample buffer in order to track the distance in which the proteins travel through the gel. If it is run for too long, the proteins will run off the bottom of the gel and it will mess up your results. Once the protein reach the bottom of the gel, the gel is stained in order to be able to see the individual bands of the different proteins. When the gel is stained, the protein distances will be able to be measured and compared. For a detailed procedure, refer to the Comparative Proteomics Kit I: Protein Profiler Module Lab Manual.

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

These steps will repeat for every enzyme concentration’s control, substrate and enzyme test tubes. The total amount of data points should be 6, 2 data points per concentration. After part one, clean the test tubes by correctly disposing of the substances in the waste bin under the fume hood and then proceed to clean with soap so that the test tubes can be used for part

Due to the range of concentrations the anticipated trend was not seen, because the enzyme did not become the limiting variable. Furthermore, from 1% to 3% the intervals were .5% instead of .25% a smaller interval would make the data more clear. This had a large impact on the yielded results as the anticipated trend was not observable. The range of the concentration should be from 0.00% to 5% with .25% intervals to make the data more clear and to see if the hypothesized trend would be observable.

Our first step was that we boiled the samples, A&M Std , & Kaleidescope Std. for 5min. in water bath, then we loaded the samples into the wells following the guide given which was as follows: 1- 10 ml of laemmli buffer, 2- 10 ml of molecular weight, 3- 10 ml of salmon, 4- 10 ml of tilapia, 5- 10 ml of catfish, 6- 10 ml of shrimp, 7- 10 ml of actin and myosin, and 8- 10 ml of the laemmli buffer. To load each sample, we used a P-200 (yellow) micropipette tip to withdraw 10 ml of each protein sample from its tube and gently transferred it into the designated well. After loading all samples, on both of the gels we then placed the lid on the tank, and insert the leads into the power supply, matching red to red and black to black. We ran the gel for 45 minutes at a constant voltage of 115V. When the gels were finished running, we discarded the buffer from the inner chamber, we released the cams, and removed the gel cassettes from the assembly. We laid each gel cassette flat on the bench with the short plate facing

The more agarose that is used and dissolved, the firmer the gel will be. Typical concentrations used are between 0.3% to 2% (Buckingham, 2012). The concentration depends on the type of analysis needed. A higher concentration of agarose, making a stiffer gel, would be used to detect smaller DNA fragments (100-3000 bp) while a lower concentration would be used for larger fragments (5000-60,000 bp). For pieces 50,000 bp and over, pulsed field electrophoresis is used where an alternating current is applied (Buckingham, 2012). A gel comb is placed into one side of the gel mold to create holes (wells) in the gel. The gel will need to cool and solidify, about an hour. The comb is then removed, which leaves the empty wells in the gel.

When picking up the gel, the gel broke in half. This could have altered the results because some of our DNA could have broken off. Although it appeared that we saved the DNA, there is a slight chance that some of the DNA could have broken off. Also, my assistants and I had difficulty measuring the agarose powder. We might have poured too much of the powder, which could have made the gel too thick. Next time, we will be more cautious when measuring the powder to have more accurate results.

Following this action, in tubes 1 and 3 15-µl of Enzyme 1 was dispensed and the same amount was dispensed for tubes 2 and 4 of Enzyme 2. The final volumes of the each of the reaction tubes had a final volume of 40-µl (Table 1). After dispensing what was needed in each of the tubes, one of the team members tapped each tube to mix all the contents together and to be sure that the contents were at the bottom of each tube. Sequentially, after all the contents were mixed and were at the bottom, the tubes were then placed in an incubator at 37°C for 45 minutes (Upadhayaya, 2016). Prior to removing the DNA samples from the incubator, the Gel for Electrophoresis had to be casted.

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.

You do not need to complete this graph if you attach the printout of the computer generated graph

However, in order to find a linear slope of a parabolic curve, the mathematical equation of distance/time^2 ( pertaining to the data as cm/s^2) is used to produce the linearization of the distance-time graph. A linear distance-time graph is the equivalent of a velocity-time graph by identifying the slope of the distance-time graph. Another best fit line (a linear trendline) was plotted within the newly charted velocity-time graph. The equation identified through the trendline was Velocity=808.578cm/s^2 (t) +44.450 cm/s. The 44.450cm/s represents the point in which the linear data crosses the y-axis as the y-intercept. Additionally, the 808.578cm/s^2 represents the slope of the velocity-time graph in which the acceleration can be

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)

The overall feedback was to state the aim and conclusion clearly, include more in-text citations and references and be more detailed in elements of experimental errors. This feedback was taken into account, as I have included more in-text citations and references, expanded on experimental errors in more detail and ensured my aim and conclusion is clear.