M15 Worksheet - Gel Electrophoresis PDF

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The stimulations are completed at level 3 on LabXchange gel electrophoresis stimulation....

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MODULE 15: GEL ELECTROPHORESIS Instructions: Answer the questions on material from the lectures. Complete the LabXChange gel electrophoresis simulation at level 3. Submit completed worksheet in Assignments, worth 20 points. 1. Ladders are run on both agarose and SDS-PAGE gels. Describe the purpose and composition of ladders for both types of gels. (2 pt) a. Agarose gel: The agarose gel is as diagnostic tool which is used to visualize fragments. The DNA fragments are separated according to their size from this gel. b. SDS-PAGE gel: The protein is separated by it based on their length qnd molecular weight in an inexpensive, easy and accurate manner. 2. In SDS-PAGE, the following chemicals are added to the protein sample before loading. (4 pt) Describe the purpose of each: a. Sodium dodecyl sulfate: It is used to set to set a condition for electrophoresis in which all the protein are separated into same size of bands. b. Beta-mercaptoethanol: it is used as a reducing agent to break the disulfide bridges. c. Bromophenol blue: Moves in the same direction of the protein that is separating, when added to the buffer it acts as a tracking dye and demarcates their leading edge. d. Glycerol: It is used to increase the sample’s density so that when the wells are loaded, they will sink. 3. In SDS-PAGE, the separating gel is pH 8.8 and the stacking gel is pH 6.8. Explain why they are different pHs and describe how they work. (2 pt) a. Stacking gel: Its purpose is to line up the protein sample that is loaded on the gek so that they can be entered in the resolving gel at the same time. b. Separating gel: The samples are separated based on their molecular weight, which is allowed by this gel. 4. Sodium dodecyl sulfate is present in the gel and running buffer, why? (1 pt) Ans: Sodium dodecyl sulfate is present in the gel and running buffer because it is used to make the proteins negatively charged and denature them. Each protein will transport to the electrophoretic field at a rate equal to its length. 5. You have learned about two different types of gels in this module: Agarose and SDS-PAGE. Which type would you use in a lab analysis for each of the following molecules? If you don’t recognize the names, look for a definition online. (1 pt) Sample Plasmid cDNA mRNA Enzyme Kinase

Gel Type ag ag ag sdsp sdsp

MODULE 15: GEL ELECTROPHORESIS Phosphorylase Myosin Insulin EcoRI restriction endonuclease

sdsp sdsp sdsp sdsp

6. Answer the following questions for the image of this protein gel.

a. How many bands are in sample 2? (1/2 pt) ANS: 2

b. What are their molecular weights? (1/2 pt) ANS: 128

c. Let’s say this gel has samples from an experiment in a molecular lab where they are studying a pathogenic virus. The scientist plans to infect mice that may or may not be resistant to infection. A specific protein with MW of 86 kDa has been implicated to have a role in a strong resistance response to the disease. She runs

MODULE 15: GEL ELECTROPHORESIS the proteins from three blood samples on the SDS-PAGE gel checking for presence of this protein. She loads a ladder in lane 1, and samples from 3 mice in lanes 2 through 4. Which lanes have samples from mice that are likely to be resistant? (1 pt) Ans: The sample 1 and 2 7. Experimental data is more likely to look like this: 1 2 3 4 5 6 7 8 9

10

11 12 13

A scientist is looking for a protein that is approximately 32 kDa in order to purify large quantities for further experiments. She loads multiple protein samples from her library of E. coli clones that may be expressing a human protein from a recombinant plasmid. This gel is a screen to choose which clone expresses the most of her desired protein. a. In this gel, number the lanes and identify the samples. Clearly describe them so an observer can easily determine which lane you’re talking about. (1 pt) Ans: 13 lanes are labeled above the picture. b. Which lanes have the best candidates? Which one would you choose for your further experiments, and why? (2 pt) Ans: Since the lanes 2, 12 and 13 are the closest to 32 kDa, I would use them.

MODULE 15: GEL ELECTROPHORESIS LABXCHANGE GEL ELECTROPHORESIS SIMULATION. Start the simulation at level 3. Level 1 is used for high school. LabXChange gel electrophoresis simulation.

1. Paste a screen capture of your predicted results here. (1/2 pt)

2. Paste your actual results here with your explanation of why the bands ended up in those relative positions. Is this what you predicted? Explain. (1 pt)

The charge and the size of fragment determines how far the bands migrate in the gel. As the mass of purple dye is more than the mass of blue dye, therefore, the purple dye migrates further in the gel even though the size is larger.

The following questions were copied from the LabXchange exercise. Choose the correct answers from the multiple choices on the website and type in the correct answers here. 3. Your lab partner put the gel into the electrophoresis box as shown below with the wells oriented towards the positive electrode. You run the same experiment as

MODULE 15: GEL ELECTROPHORESIS conducted in this simulation with the S1, S2 and S3 samples. How would this impact your results? (1/2 pt) Ans: It would result in an empty gel because the dyes will move toward the positive end and then it will run off the gel’s surface.

4. Why might you run gel electrophoresis with an agarose gel like the one pictured below where the wells are in the center of the gel? (1/2 pt) Ans:

The wells in the middle gives the option to the molecules to travel in both the direction. So, if the molecules of unknown charges have to be separated, then electrophoresis with an agarose gel like this is run.

5. Which is the best explanation for why the S1, S2, and S3 samples showed different numbers of bands? (1/2 pt) Ans: These showed different number of bands because each band is collection of a particular size of molecules and also, the S2 solution included the mixture of dye molecules of various sizes. 6. What would happen if you increased the length of time a gel is run during a gel electrophoresis experiment? (1/2 pt) Ans: The sample would run out of the bottom of the gel if the length of time is increased to run electrophoresis. 7. If you use gel electrophoresis to separate samples of DNA and get bands in different positions on the gel, what do you know about the molecules in those bands? (1/2 pt) Ans: Some of the fragments have different or longer DNA sequence and the molecules are of different sizes.

MODULE 15: GEL ELECTROPHORESIS 8. How confident do you feel about using the technique you learned in a real lab? What is your opinion of this lab simulation? (1 pt) Ans: I am not sure about how I would do in real lab because learning online and doing in person are different things for me. However, these stimulations really explained me how to do stuff in the lab and for sure help me to make less mistakes....