The MiniOne TM Reagent Kit: Crime Scene Invesitigation

Transcription

1 The MiniOne TM Reagent Kit: Crime Scene Invesitigation Cat#: M3005

2 2 Crime Scene Investigation Reagent Kit Components Other Required Materials Laboratory Safety Table of Contents Page Experiment Procedures Teacher s Guide Student Worksheet Additional Resources Ordering Consumables

3 Crime Scene Investigation 3 Reagent Kit Components Ready-to-Load Samples (please see scenario) DNA V (Victim) DNA S1 (Suspect 1) DNA S2 (Suspect 2) DNA S3 (Suspect 3) DNA H (Hair 2) Supplies 10x 1% Agarose GreenGel in-a-cup Concentrated 20x Sodium Borate (20x SB) running buffer Microcentrifuge tubes Micropipette tips MiniOne TM Electrophoresis System Running tank Carriage base Photo hood 42V Power Supply Conical flask MiniOne TM Casting System Casting stand Gel trays Gel combs Required Materials Other Materials Micropipette Microwave Oven Digital Camera or Cell Phone Camera Laboratory Safety 1. Exercise caution when heating and/or melting reagents. 2. Exercise caution when working with electrical equipment. 3. Gloves and eye protection should be used whenever possible, as a part of good laboratory practice. 4. Always wash hands thoroughly after handling biological materials or reagents.

4 4 Teacher s Guide Crime Scene Investigation Teacher s Guide Note: This guide is provided to give sample answers to questions and to explain certain steps in the protocol. There may be other correct answers that are not included. Since there are variations in data, students should carefully analyze their data and justify their conclusions based on them. Experiment Objective To develop an understanding of electrophoresis principles. To understand and use forensic science principles to analyze data and to determine a probable conclusion. Scenario Dr. Phillip Ward, a prominent physician, was found dead in his apartment. The cause of death was apparently from potassium cyanide ingestion. Dr. Ward lives alone and does not own any pets. The police suspect that this may be a suicide case, but they are not sure at this time. Your team of forensic specialists had been assigned to this case and upon examining the crime scene, you found several pieces of evidence. Traces of potassium cyanide were found in Dr. Ward s coffee cup. When examining the cup, you lifted some fingerprints. On floor of the kitchen, you found a piece of hair that did not match the color of the victim s hair. Lastly, among the trash in the apartment, you found a bag with white powder, which contains traces of potassium cyanide. You also found an eyelash stuck on the bag. There was just enough of the hair root on the second hair sample that you were able to extract another DNA sample. You also took light microscope images of both hair samples. The police want information based on the physical evidence as soon as possible. Does the evidence support a suicide theory, or could this be a murder case? Is there evidence that someone other than Dr. Ward was in his apartment? Lesson Plan Time Line Day 1- Day 2- Day 3- Introduce scenario and crime scene. Hair analysis (30-45mins depending on background) Fingerprint analysis (30-45mins depending on background). Pour gels (30 mins). DNA analysis (30 mins) Note: If students have sufficient background already, hair and fingerprint analysis can be combined in one day. Hair and fingerprint make the story more interesting but either are not essential to solving the case. DNA analysis can be sufficient. The teacher can pour all the gels for students to reduce the time needed.

5 Crime Scene Investigation Teacher s Guide 5 Crime Scene Evidence DNA Samples: DNA V (Victim) DNA S1 (Suspect 1) Fingerprints: Victim Fingerprint 1 on coffee cup Fingerprint 2 on coffee cup Hair Samples: Victim Microscope Image of Hair 1 Microscope Image of Hair 2 Other: Coffee cup Bag with white powder

6 6 Teacher s Guide Crime Scene Investigation Suspects The police have been identifying and investigating suspects in the possible murder of Dr. ward. They have narrowed the list of suspects to three people. The police also took samples of their DNA. (S1) Dr. Caroline Powell: Dr. Powell is a colleague of Dr. Ward s and they were co-owners of their joint practice for the past ten years. Employees remarked that Dr. Powell seemed to resent Dr. Ward for his success and felt eclipsed by him. Dr. Powell had been heard arguing with Dr. Ward recently. Now that Dr. Ward is dead, their practice belongs solely to her. She does not have an alibi. Dr. Powell owns a cat. (S2) Mrs. Julie Fischer: A neighbor of Dr. Ward s in the same apartment building. Mrs. Fischer owns a Pomeranian. Other neighbors said that Dr. Ward had complained about Mrs. Fischer s dog barking too loudly and had threatened to report her to their building management. Mrs. Fischer claims that she was eating out with her husband that night. Her husband s version of the story is slightly different. (S3) Miss Lauren White: The receptionist at Dr. Ward s practice. She is also the girlfriend of Dr. Ward s nephew, Oliver. Oliver was Dr. Ward s only living relative and was the beneficiary of his life insurance policy. Miss White claims that she was at the movies that night, but no one can confirm her alibi. Miss White does not own any pets, but Oliver owns a cat. Suspect Dr. Powell Mrs. Fischer Miss White DNA Sample S1 S2 S3 Fingerprint Hair Sample

7 Crime Scene Investigation Teacher s Guide 7 Hair Analysis Hair can be important pieces of evidence during an investigation. Since hair is easily transferred during physical contact, hair samples can associate a person to a crime scene. What would you do if you were given hair and asked to determine if it came from a suspect? One of the first questions a scientist will ask is whether the hair came from an animal or a human. If the hair is of animal origin, they will examine its morphology with a light microscope to determine the species. However, it is not possible to distinguish the hair between individual animals. If the hair is from a human, it may be possible to identify individuals. Although the morphology of human hair is shared across individuals in the population, there are certain physical characteristics (such as arrangement, distribution or appearance) that skilled scientists can use to distinguish between people. Cuticle Cortex Medulla Physical Structure of Hair: Cuticle outer coating, made of overlapping scales Cortex main body of hair, is protein-rich and contains pigment granules Medulla central core, may be absent, fragmented or distinctly structured As you examine these images from different species, think about how they differ and which characteristics you could use to determine identity. Fill in the chart below. Species Microscope Image Description Human Dog Cat Rabbit Cuticle - Cortex - Medulla - Cuticle - Cortex - Medulla - Cuticle - Cortex - Medulla - Cuticle - Cortex - Medulla - Not prominent, smooth Pigment dense, clustered Amorphous, fragmented Not prominent, smooth Ovoid bodies present Continuous, wide Spiny, prominent Not prominent Continuous, uniserial ladder Not prominent, smooth Not prominent Continuous, multiserial ladder

8 8 Teacher s Guide Crime Scene Investigation 1. Based on the microscope images of the hair samples and the chart above, what is the species of Hair 1? What characteristics from the hair sample led you to your conclusions? Hair 1 is from a cat. Spiny cuticle, uniserial ladder in the medulla 2. Based on the microscope images of the hair samples and the chart above, what is the species of Hair 2? What characteristics from the hair sample led you to your conclusions? Hair 2 is from a human. Smooth/not prominent cuticle, dense pigment distribution in the cortex, and amorphous/fragmented medulla. 3. Based on the species and color of the hair samples, can you associate any of the suspects with the crime scene? Explain your reasoning. Hair 2 is from a human and has a dark brown color. The suspects who have darker hair are S2 (Mrs. Fischer) and S3 (Miss White). S1 (Dr. Powell) has light brown hair and hair 2 would not be from her. Hair 1 is from a cat and would not be directly from any of the suspects. However, all three of the suspects have pets or were potentially in contact with pets. S3 does not own a cat, but her boyfriend does, and the cat hair could have fallen off from her clothes. S1 owns a cat as well and could have contributed the hair. S2 owns a dog, so this suspect is less likely to have contributed hair 1 to the crime scene. 4. During the crime scene investigation, you were able to find enough of the hair root on Hair 2 to do an analysis. What analysis would you do to help you further pinpoint the murderer? How might you go about doing that? The follicle attached to Hair 2 includes cells that contain DNA. You can do a DNA extraction of nuclear DNA. After extracting DNA, you can do one of several techniques. One is the RFLP method and there are also PCR based methods. The non-coding portions of human DNA often have many distinct repeating patterns, as a result of accumulated mutations over generations. Both techniques are based on this concept. For RFLP, these regions are cut with restriction enzymes and when separated by gel electrophoresis, they result in different banding patterns. For PCR based methods, certain short repeats in the DNA sample are amplified and then result in a mixture of differently sized DNA fragments. These results are also separated on a gel. For both, the more similar the patterns, the higher the probability that samples are the same individuals or are related.

9 Crime Scene Investigation Teacher s Guide 9 Fingerprint Analysis Fingerprints are still considered as one of the best types of physical evidence for various reasons. Fingerprints are more unique than DNA. Although identical twins share genetic information, they do not share the same fingerprints. While fingerprint formation has some genetic basis, they are made when pressure on the skin layers of the fetus experience pressure, resulting ridges patterns. These patterns do not change when a person ages, although the prints can acquire scars or get worn down. Each ridge contains pores, which are connected to sweat glands. When you touch a surface, you leave fingerprints because of this sweat. Scientists can pinpoint minute differences in fingerprint patterns to distinguish between them. You will take on this role today by determining if any of the suspects could have left fingerprints at the crime scene. Types of Fingerprint Patterns: 1. Identify the fingerprint patterns for each of the suspects and the victim. S1 (Dr. Powell): Right pocket loop, S2 (Mrs. Fischer): Right loop, S3 (Miss White): Whorl, Victim: Whorl 2. What fingerprint patterns can you pick out in the fingerprints on the coffee cup? On the bag? Fingerprint 1: appears to be a mix of 2 or more prints. Perhaps a whorl or a type of loop, Fingerprint 2: Whorl 3. Based on the fingerprint analysis, which suspect is more suspicious, if any? Why? For Fingerprint 1, the mixed prints may result from the victim and/or the murderer touching the cup. Whorls are most prominent in this print, which may point to S3 as a possibility. Fingerprint 2 does not match the victim s fingerprint. The suspect who also has a whorl fingerprint is S3, again pointing to her having contact with the coffee cup and possibly the one responsible for adding potassium cyanide.

10 10 Teacher s Guide Crime Scene Investigation DNA Analysis While each individual is unique, their very DNA holds clues to their history and heritage. These clues are held in the non-coding regions of DNA, where random mutations are more likely to come up. As these changes usually do not affect essential genes to survival, the variation is passed along from parent to offspring and over generations, the DNA region has a distinct pattern. The technique of DNA fingerprinting takes advantage of this concept to access the similarity between certain individuals. Restriction enzymes recognize particular DNA sequences and are used in DNA fingerprinting to create differently sized pieces of DNA. When separated by gel electrophoresis, we can create a banding pattern for each individual s DNA sample. The more similar banding patterns, the higher the probability that individuals are related. This technique is now used in many areas, from crime scenes to paternity cases for determining identity or relationships. Note: This kit provides enough materials to run 10 Before the Lab experiments. The following suggested preparation steps assume that you are preparing to run all 10 experiments. Prepare Running Buffer 1. The kit provides 100 ml of concentrated 20x Sodium Borate (20x SB) buffer. Dilute 1 volume of the concentrated 20x SB buffer with 19 volumes of DI water to get a 1x SB working concentration. Estimate that each experiment will need 125 ml of running buffer, plus extra in case of spills. Example calculation: (for 10 runs plus extra) Final volume: 2000 ml 20x SB needed: 2000 ml / 20 = 100 ml DI water needed: 2000 ml 100 ml = 1900 ml 2. Following the calculation, dilute 100 ml of 20x SB with 1900 ml of DI water to get 2 L of 1x SB for running buffer. 3. Optional: Aliquot 125 ml of diluted SB running buffer for each group of students before class starts, using the provided conical flasks. Prepare Sample Aliquots 1. The kit provides a total of 120 μl for each sample. 2. With the provided microcentrifuge tubes, aliquot 12 μl per sample for each group. There are a total of 5 DNA samples. Other Materials (Each group will need) 1 MiniOne Casting System 1 MiniOne Electrophoresis System 1 Agarose GreenGel in-a-cup 5 sample aliquots 125 ml of diluted SB running buffer, in the conical flask 1 micropipette & 5 pipette tips

11 Crime Scene Investigation Teacher s Guide 11 Experiment Overview 1 Peel & microwave 30 sec 2 Cast the gel 3 Add running buffer & gel to buffer tank. Place tank into carriage 4 Load samples - 5 Run the gel 6 Record an image +

12 12 Teacher s Guide Crime Scene Investigation Prelab Questions 1. What is DNA and what does it do? Deoxyribonucleic acid, makes you who you are, codes for proteins, etc. 2. What are chromosomes and how many copies do you have in each of your cells? Who did they come from? Chromosomes are DNA that is packaged with proteins, so that they will fit into the nucleus of a cell. You have 2 copies of each of the 22 autosomal (non-sex determining) chromosomes and either 2 X s, if you are female, or an X and Y, if you are male. One entire set of 23 chromosomes (22 autosomal+1 sex chromosome) came from your mother and an entire other set from your father, totaling 46 chromosomes in every cell of the body. 3. How much DNA do you share with each of your parents? How much DNA do you share with your siblings? Parents: 50%, a set of 23 chromosomes from each. Siblings: Between 0% and 100% (baring in mind that all humans are 99.9% the same on a DNA level, these numbers are DNA that you received directly from your parents and that your siblings received directly from your parents.) 4. What are some characteristics or properties of DNA? Double helix, double stranded, negatively charged, colorless, unique sequence in each individual

13 Crime Scene Investigation Teacher s Guide 13 Part I: Electrophoresis Well Sample Name Loading Materials 1 MiniOne Casting System 1 MiniOne Electrophoresis System 1 Agarose GreenGel in-a-cup 5 samples Diluted SB running buffer 1 micropipette & 5 pipette tips Photo hood Volume 1 Victim (V) 10 μl 2 Suspect 1 (S1) 10 μl 3 Suspect 2 (S2) 10 μl 4 Suspect 3 (S3) 10 μl 5 DNA from Hair 2 (H) 10 μl Procedure 1. Assemble MiniOne casting system. Place the casting stand on a level surface. Place gel trays in the two cavities of the casting stand and insert the comb into the slots at the top of the casting stand, such that the 6 well side is facing down. 2. Partially peel away the seal of a Agarose GreenGel in-a-cup and microwave for 30 secs. Slowly pour the hot gel solution into a gel tray. Check to see that there are no air bubbles in the gel solution. Let the agarose gel harden and turn opaque, approximately mins. (DO NOT touch or move your gel until time is up.) Note: If melting multiple cups together, still microwave for 30 secs. Then check that each cup is completely melted. If not, add time in 5 sec increments until fully melted. Air bubbles in the gel solution, especially near the wells, can make it hard for DNA to migrate evenly (if there are bubbles, you can remove them with a pipette tip or a toothpick). If the gel is disturbed before it fully hardens, the gel can break or solidify unevenly, making it harder for DNA to migrate evenly. 3. Meanwhile, assemble the MiniOne electrophoresis system. With the included power supply, plug the carriage into the wall outlet. Leave the clear running tank separate from the carriage for now. Place the platform into the tank if it is not already installed. (DO NOT turn the power on yet.) 4. After the gel has hardened, carefully remove the comb. Then, remove the gel with the gel tray from the casting stand. Note: If you are having trouble removing the comb, pour some SB buffer on top of the gel and then completely pull the comb out. 5. Place the gel with the gel tray inside the running tank. Make sure that the wells are towards the negative end. (DO NOT turn the power on yet.)

14 14 Teacher s Guide Crime Scene Investigation 6. Measure out 125 ml of diluted SB running buffer and pour slowly into one side of the tank. The buffer will flow under the gel, towards the other side, and remove any air bubbles. Place the tank into the carriage, so that it sits level. 7. Turn the low intensity lights on by pressing the button on the carriage to help visualize the wells. Load your DNA samples into the wells, using 10 μl per well and changing to a new pipette tip each time. Remember to keep track of which samples you loaded in which wells. 8. Run that gel! While being careful not to bump your gel too much, make sure that the power cord is completely plugged in and place the photo hood on the carriage. Then press the power button. The green LED next to the power button should turn on. Note: The power will not turn on if 1) the tank is not appropriately inside the carriage, 2) there is no buffer in the tank or 3) the photo hood is not on on the carriage. 9. The blue light allows you to see the DNA as it is running, and the orange photo hood helps filter excess light to improve contrast. If you remove the photo hood during the run, power will shut off immediately. Remember to replace the hood and press the power button to resume run. 10. Allow the gel to run approximately mins or until DNA separation is sufficient. After your run is complete, turn off the power by pressing button. Use the photo hood and either your cell phone or camera to take a picture of the DNA. (Be sure to wipe off condensation from the inside of the hood, if any). Part II: Results What does your gel look like? Record an image of the gel. Sample Results Note: DNA is negatively charged, so to move the DNA into the gel with electricity, the DNA needs to be loaded on the negative side. It will then move towards the positive. What do you think the SB buffer is for? SB conducts electricity and completes the circuit so that DNA can be separated. V S1 S2 S3 H

15 Crime Scene Investigation Teacher s Guide 15 Part III: Analyze Your Data 1. What electrical charge did your samples carry? How do you know? All the samples have components that are negatively charged. You know because they all move towards the positive end when the power is turned on. If there were anything positively charged, then they would migrate towards the negative end and can run out the top of the gel. 2. According to the results on the gel and other pieces of evidence from the crime scene, who was responsible for the death of Dr. Ward? How did you come to this conclusion? The eyelash stuck on the bag with potassium cyanide is a crucial piece of information because only a person who had handled the poison could have left it. If the DNA of the eyelash had been Dr. Ward s, then this points towards suicide. However, if the DNA had been any of the suspects, then either they were responsible or were an accomplice. Students should carefully analyze their data and come to a conclusion. They can also use the information inferred from the fingerprints and hair samples to present their case. If their data is inconclusive, they should explain that they would need to redo the experiment.

16 16 Student Worksheet Crime Scene Investigation Student Worksheet Experiment Objective To develop an understanding of electrophoresis principles. To understand and use forensic science principles to analyze data and to determine a probable conclusion. Scenario Dr. Phillip Ward, a prominent physician, was found dead in his apartment. The cause of death was apparently from potassium cyanide ingestion. Dr. Ward lives alone and does not own any pets. The police suspect that this may be a suicide case, but they are not sure at this time. Your team of forensic specialists had been assigned to this case and upon examining the crime scene, you found several pieces of evidence. Traces of potassium cyanide were found in Dr. Ward s coffee cup. When examining the cup, you lifted some fingerprints. On floor of the kitchen, you found a piece of hair that did not match the color of the victim s hair. Lastly, among the trash in the apartment, you found a bag with white powder, which contains traces of potassium cyanide. You also found an eyelash stuck on the bag. There was just enough of the hair root on the second hair sample that you were able to extract another DNA sample. You also took light microscope images of both hair samples. The police want information based on the physical evidence as soon as possible. Does the evidence support a suicide theory, or could this be a murder case? Is there evidence that someone other than Dr. Ward was in his apartment?

17 Crime Scene Investigation Student Worksheet 17 Crime Scene Evidence DNA Samples: DNA V (Victim) DNA S1 (Suspect 1) Fingerprints: Victim Fingerprint 1 on coffee cup Fingerprint 2 on coffee cup Hair Samples: Victim Microscope Image of Hair 1 Microscope Image of Hair 2 Other: Coffee cup Bag with white powder

18 18 Student Worksheet Crime Scene Investigation Suspects The police have been identifying and investigating suspects in the possible murder of Dr. ward. They have narrowed the list of suspects to three people. The police also took samples of their DNA. (S1) Dr. Caroline Powell: Dr. Powell is a colleague of Dr. Ward s and they were co-owners of their joint practice for the past ten years. Employees remarked that Dr. Powell seemed to resent Dr. Ward for his success and felt eclipsed by him. Dr. Powell had been heard arguing with Dr. Ward recently. Now that Dr. Ward is dead, their practice belongs solely to her. She does not have an alibi. Dr. Powell owns a cat. (S2) Mrs. Julie Fischer: A neighbor of Dr. Ward s in the same apartment building. Mrs. Fischer owns a Pomeranian. Other neighbors said that Dr. Ward had complained about Mrs. Fischer s dog barking too loudly and had threatened to report her to their building management. Mrs. Fischer claims that she was eating out with her husband that night. Her husband s version of the story is slightly different. (S3) Miss Lauren White: The receptionist at Dr. Ward s practice. She is also the girlfriend of Dr. Ward s nephew, Oliver. Oliver was Dr. Ward s only living relative and was the beneficiary of his life insurance policy. Miss White claims that she was at the movies that night, but no one can confirm her alibi. Miss White does not own any pets, but Oliver owns a cat. Suspect Dr. Powell Mrs. Fischer Miss White DNA Sample S1 S2 S3 Fingerprint Hair Sample

19 Crime Scene Investigation Student Worksheet 19 Hair Analysis Hair can be important pieces of evidence during an investigation. Since hair is easily transferred during physical contact, hair samples can associate a person to a crime scene. What would you do if you were given hair and asked to determine if it came from a suspect? One of the first questions a scientist will ask is whether the hair came from an animal or a human. If the hair is of animal origin, they will examine its morphology with a light microscope to determine the species. However, it is not possible to distinguish the hair between individual animals. If the hair is from a human, it may be possible to identify individuals. Although the morphology of human hair is shared across individuals in the population, there are certain physical characteristics (such as arrangement, distribution or appearance) that skilled scientists can use to distinguish between people. Cuticle Cortex Medulla Physical Structure of Hair: Cuticle outer coating, made of overlapping scales Cortex main body of hair, is protein-rich and contains pigment granules Medulla central core, may be absent, fragmented or distinctly structured As you examine these images from different species, think about how they differ and which characteristics you could use to determine identity. Fill in the chart below. Species Microscope Image Description Human Dog Cat Rabbit Cuticle - Cortex - Medulla - Cuticle - Cortex - Medulla - Cuticle - Cortex - Medulla - Cuticle - Cortex - Medulla -

20 20 Student Worksheet Crime Scene Investigation 1. Based on the microscope images of the hair samples and the chart above, what is the species of Hair 1? What characteristics from the hair sample led you to your conclusions? 2. Based on the microscope images of the hair samples and the chart above, what is the species of Hair 2? What characteristics from the hair sample led you to your conclusions? 3. Based on the species and color of the hair samples, can you associate any of the suspects with the crime scene? Explain your reasoning. 4. During the crime scene investigation, you were able to find enough of the hair root on Hair 2 to do an analysis. What analysis would you do to help you further pinpoint the murderer? How might you go about doing that?

21 Crime Scene Investigation Student Worksheet 21 Fingerprint Analysis Fingerprints are still considered as one of the best types of physical evidence for various reasons. Fingerprints are more unique than DNA. Although identical twins share genetic information, they do not share the same fingerprints. While fingerprint formation has some genetic basis, they are made when pressure on the skin layers of the fetus experience pressure, resulting ridges patterns. These patterns do not change when a person ages, although the prints can acquire scars or get worn down. Each ridge contains pores, which are connected to sweat glands. When you touch a surface, you leave fingerprints because of this sweat. Scientists can pinpoint minute differences in fingerprint patterns to distinguish between them. You will take on this role today by determining if any of the suspects could have left fingerprints at the crime scene. Types of Fingerprint Patterns: 1. Identify the fingerprint patterns for each of the suspects and the victim. 2. What fingerprint patterns can you pick out in the fingerprints on the coffee cup? On the bag? 3. Based on the fingerprint analysis, which suspect is more suspicious, if any? Why?

22 22 Student Worksheet Crime Scene Investigation DNA Analysis While each individual is unique, their very DNA holds clues to their history and heritage. These clues are held in the non-coding regions of DNA, where random mutations are more likely to come up. As these changes usually do not affect essential genes to survival, the variation is passed along from parent to offspring and over generations, the DNA region has a distinct pattern. The technique of DNA fingerprinting takes advantage of this concept to access the similarity between certain individuals. Restriction enzymes recognize particular DNA sequences and are used in DNA fingerprinting to create differently sized pieces of DNA. When separated by gel electrophoresis, we can create a banding pattern for each individual s DNA sample. The more similar banding patterns, the higher the probability that individuals are related. This technique is now used in many areas, from crime scenes to paternity cases for determining identity or relationships. Prelab Questions 1. What is DNA and what does it do? 2. What are chromosomes and how many copies do you have in each of your cells? Who did they come from? 3. How much DNA do you share with each of your parents? How much DNA do you share with your siblings? 4. What are some characteristics or properties of DNA?

23 Crime Scene Investigation Student Worksheet 23 Experiment Overview 1 Peel & microwave 30 sec 2 Cast the gel 3 Add running buffer & gel to buffer tank. Place tank into carriage 4 Load samples - 5 Run the gel 6 Record an image +

24 24 Student Worksheet Crime Scene Investigation Part I: Electrophoresis Materials 1 MiniOne Casting System 1 MiniOne Electrophoresis System 1 Agarose GreenGel in-a-cup 5 samples Diluted SB running buffer 1 micropipette & 5 pipette tips Well Sample Name Loading Volume 1 Victim (V) 10 μl 2 Suspect 1 (S1) 10 μl 3 Suspect 2 (S2) 10 μl 4 Suspect 3 (S3) 10 μl 5 DNA from Hair 2 (H) 10 μl Photo hood Procedure 1. Assemble MiniOne casting system. Place the casting stand on a level surface. Place gel trays in the two cavities of the casting stand and insert the comb into the slots at the top of the casting stand, such that the 6 well side is facing down. 2. Partially peel away the seal of a Agarose GreenGel in-a-cup and microwave for 30 secs. Slowly pour the hot gel solution into a gel tray. Check to see that there are no air bubbles in the gel solution. Let the agarose gel harden and turn opaque, approximately mins. (DO NOT touch or move your gel until time is up.) 3. Meanwhile, assemble the MiniOne electrophoresis system. With the included power supply, plug the carriage into the wall outlet. Leave the clear running tank separate from the carriage for now. Place the platform into the tank if it is not already installed. (DO NOT turn the power on yet.) 4. After the gel has hardened, carefully remove the comb. Then, remove the gel with the gel tray from the casting stand. 5. Place the gel with the gel tray inside the running tank. Make sure that the wells are towards the negative end. (DO NOT turn the power on yet.) 6. Measure out 125 ml of diluted SB running buffer and pour slowly into one side of the tank. The buffer will flow under the gel, towards the other side, and remove any air bubbles. Place the tank into the carriage, so that it sits level. 7. Turn the low intensity lights on by pressing the button on the carriage to help visualize the wells. Load your DNA samples into the wells, using 10 μl per well and changing to a new pipette tip each time. Remember to keep track of which samples you loaded in which wells.

25 Crime Scene Investigation Student Worksheet 25 Procedure (cont.) 8. Run that gel! While being careful not to bump your gel too much, make sure that the power cord is completely plugged in and place the photo hood on the carriage. Then press the power button. The green LED next to the power button should turn on. 9. The blue light allows you to see the DNA as it is running, and the orange photo hood helps filter excess light to improve contrast. If you remove the photo hood during the run, power will shut off immediately. Remember to replace the hood and press the power button to resume run. 10. Allow the gel to run approximately mins or until DNA separation is sufficient. After your run is complete, turn off the power by pressing button. Use the photo hood and either your cell phone or camera to take a picture of the DNA. (Be sure to wipe off condensation from the inside of the hood, if any).

26 26 Student Worksheet Crime Scene Investigation Part II: Results What does your gel look like? Record images of the gel. Part III: Analyze Your Data 1. What electrical charge did your samples carry? How do you know? 2. According to the results on the gel and other pieces of evidence from the crime scene, who was responsible for the death of Dr. Ward? How did you come to this conclusion?

27 Crime Scene Investigation 27 Additional Resources Hair Analysis: Background Information & Images research/2004_01_research01b.htm research/2004_03_research02.htm DNA Testing: Introduction

28 28 Crime Scene Investigation Call (858) or (800) Ordering Consumables Cat # Item Name Description M3001 Electrophoresis 101 Kit Basics of electrophoresis, for 10 groups. M3002 M3003 How to Pipette & Practice Gel Loading Kit PTC- To Taste or Not To Taste Kit Learn to pipette and practice fine motor control, for 20 groups. Relate genetics and electrophoresis as a tool, for 10 groups. M3004 DNA Fingerprinting Kit Examine genetic variations, for 10 groups. M3005 M3102 M3101 Crime Scene Investigation 1% Agarose GreenGels in-a-cup Concentrated 20x Sodium Borate Buffer Intro to forensic science, including hair, fingerprint & DNA analysis, for 10 groups Easily cast gels to adapt your experiments, 10 gels/pk. Just dilute and it is ready to use. M3104 MiniOne Marker Load next to samples for easy comparison 5 bands, 500 μl for 50 loads M3107 M3108 M3109 M3110 M3111 M ml Microcentrifuge Tubes, Clear 0.6 ml Microcentrifuge Tubes, Rainbow 1.7 ml Microcentrifuge Tubes, Clear 1.7 ml Microcentrifuge Tubes, Rainbow μl, Micropipette Tips, Clear 1-10 μl, Micropipette Tips, Clear For sample storage, 200/pk For sample storage, 200/pk For sample storage, 200/pk For sample storage, 200/pk For use with micropipette, non-sterile, 250/pk For use with micropipette, non-sterile, 250/pk (858)