Lab 9 Gel Electrophoresis Lab

Lab 9 Gel Electrophoresis Lab
Instructions
⦁ All work needs to be your own. This is the most important instruction. Evidence this work is not your own can lead to a zero and a report to the Dean of Students for academic dishonesty. It is not worth it. Your answers do not have to be perfect to earn an excellent grade. If you are having trouble, tell me before the deadline. It is my job to help you work through this material.
⦁ Put your answers in bold or highlight.
⦁ To help you find the questions you need to answer, those questions begin with red highlight.
⦁ Information from this lab will be tested on Exam 3 Life’s Information.

Part 1 of 5: Online Gel Electrophoresis
⦁ Go to this website http://learn.genetics.utah.edu/ or click google “learn genetics Utah.”
⦁ Click on the tab towards the bottom right of the home page for “Virtual Labs” and then click on “Gel Electrophoresis.”
⦁ If you don’t have flash player for this animated virtual lab, you can watch this video of the lab instead: https://youtu.be/lRHB9LIxkCI

Answer the following questions as you work your way through the lab.
⦁ What is your “job”?
⦁ What does gel electrophoresis do?
⦁ What is the “gel”?
⦁ Why is the gel like a sponge?
⦁ Where is the DNA placed?
⦁ What does the “electrophoresis” do?
⦁ Which strands move more quickly through the holes? Short strands or long strands?
⦁ Which strands will move farther down the gel over time? Short strands or long strands?
⦁ Why do strands of the same size get grouped together?
⦁ Why is gel stained?
⦁ What does the DNA look like in a stained gel?
⦁ What is agarose?
⦁ Why is the buffer salt water?
⦁ What happens to the agarose when heated in the microwave?
SET UP THE GEL.
⦁ What is the purpose of the comb?
⦁ What is the purpose of the purple loading buffer?
⦁ What charge does DNA have?
⦁ What charge will be near the wells with DNA? (Hint: black)
⦁ What charge will be on the other end of the gel? (Hint: red)
⦁ Which strands migrate farther? Short or long
⦁ After running the gel, what do we have to do before we can see the DNA bands?
⦁ What is the last step of the lab?
⦁ The DNA that you ran on the gel produced three bands. By comparing their position to the “DNA Size Standard” bands you can estimate the size of the DNA in the sample bands. Write the estimated size of the DNA in the bands below.

Top of Gel (- Charge)

Part 2 of 5: Digesting DNA
Digesting DNA before gel electrophoresis, requires the incubation of the extracted DNA with restriction enzymes. Restriction enzymes naturally occur in bacteria. The restriction enzyme Sma1 (first restriction enzyme discovered in the bacteria Serratia marcescens.) cuts the following sequence
5’-CCCGGG-3’
3’-GGGCCC-5’

⦁ Imagine you incubated the following DNA sequence with the restriction enzyme Sma1. Indicate where the Sma1 enzyme would cut the DNA by highlighting the sequence or putting it in bold.

5’-AACGGCCTATTAACCCGGGCCGAGAATTTCAAATGCCCGGGTATTCCCCGGG-3’
3’-TTGCCGGATAATTGGGCCCGGCTCTTAAAGTTTACGGGCCCATAAGGGGCCC-5’

⦁ If you were to digest a linear piece of DNA like the one above in three places, how many fragments would you generate?

Part 3 of 5: Analyzing Gels
When DNA fragments are run on a gel, fragments of the same size move at the same rate. They move together forming a band the same shape as the well where they were loaded. In addition, smaller fragments move faster than larger fragments. Bands that move farther down the gel are made of smaller fragments. See the example below for lane 1. Then move the bands to show the appearance of Lane 2 and Lane 3.

⦁ Lane 1: Fragments of size 90 bp, 50 bp, 35 bp
⦁ Lane 2: Fragments of size 75 bp, 10 bp
⦁ Lane 3: Fragments of 90 bp, 60 bp, 25 bp, 10 bp

Negative charge (-)

Size
Guide

Positive Charge (+)

⦁ Use the figure to answer the following question.

The segment of DNA shown in the figure has restriction enzyme cutting sites I and II, which create restriction fragments A, B, and C. Which of the gels produced by electrophoresis best represents the separation and identity of these fragments? Highlight the correct letter or put it in bold: A, B, C, D
A) C)

B) D)

Part 4 of 5: Crime Scene Analysis
DNA profiling can be used to identify criminals. In the example below, DNA was recovered from a crime scene, and there are two individuals who are suspected of the crime. A blood sample is taken from the crime scene, suspect 1 and suspect 2. The DNA is extracted from blood cells, digested, and then run on a gel. The DNA was digested with two different restriction enzymes. The resulting pattern is illustrated below.
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6
____ ____ ____
____ ____ ____
____
____ ____ ____
____ ____ ____

Lane DNA Sample
⦁ Lane 1: DNA sample from crime scene cut with Restriction Enzyme EcoR1
⦁ Lane 2: DNA sample from crime scene cut with Restriction Enzyme Sma1
⦁ Lane 3: DNA sample from Suspect 1 cut with Restriction Enzyme EcoR1
⦁ Lane 4: DNA sample from Suspect 1 cut with Restriction Enzyme Sma1
⦁ Lane 5: DNA sample from Suspect 2 cut with Restriction Enzyme EcoR1
⦁ Lane 6: DNA sample from Suspect 2 cut with Restriction Enzyme Sma1
Compare the DNA banding patterns collected from the crime scene with those taken from the suspects. Look for similarities in enzyme cutting patterns and the subsequent bands produced on the gel.
⦁ Which of the suspect’s DNA matches the DNA found at the crime scene? Why do you think so?
⦁ Could these DNA samples have been distinguished from one another if only Restriction Enzyme Eco R1 (lanes 1, 3, and 5) had been used? Why or why not?

Part 5 of 5: Paternity Test
Molecular biology has also found a use for restriction enzymes in determining the parents of a child. Until recently, blood type was used as an indicator of whether an individual could be the parent of a child but this technique has limitations. Blood type will only exclude individuals, but it will not identify them. DNA profiling can positively identify an individual. In standard genetic identification techniques, the most commonly used patterns of DNA bands should be the same for only 1 in 30 billion individuals. The world’s population is in excess of 7 billion.

The idea in testing for the biological parents is to obtain DNA from the child, mother and possible fathers and digest it with the same restriction enzymes and then separate the fragments of DNA on a gel to create a unique pattern of bands. Because a child’s DNA is a composite of its parents’ DNA, half their DNA is from the father and half their DNA is from the mother, it is possible to examine the band pattern and identify the parents. You would look for a banding pattern in the father and mother that is also found in the child’s DNA.

Continue to next page.

The gel below shows a hypothetical situation in which the father of the child is not known conclusively. Examine the pattern of bands and answer the question.
Lane 1 Lane 2 Lane 3 Lane 4
____ ____
____ ____
____ ____ ____
____ ____
____ ____

Lane DNA Sample
⦁ Lane 1: Mother’s DNA
⦁ Lane 2: Child’s DNA
⦁ Lane 3: Father 1’s DNA
⦁ Lane 4: Father 2’s DNA

1. Based on the DNA banding pattern, who is the likely father of the child? Why do you think so?

 
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