PCR Amplification of DNA for Fingerprinting

Transcription

1 The Biotechnology Education Company 130 EDVO-Kit # PCR Amplification of DNA for Fingerprinting Storage: Store the entire experiment at room temperature EXPERIMENT OBJECTIVE: The objective of this experiment is to develop a basic understanding of DNA Fingerprinting. Students will analyze PCR reactions obtained from different suspects and compare them to a crime scene sample. All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. The Biotechnology Education Company EDVOTEK

2 2 Table of Contents Page Experiment Components 3 Experiment Requirements 3 Background Information 4 Experiment Procedures Experiment Overview 7 Agarose Gel Preparation 8 Sample Delivery (Gel Loading) 12 Conducting Agarose Gel Electrophoresis 13 Staining and Visualization of DNA: InstaStain Methylene Blue 16 Staining and Visualization of DNA: Methylene Blue Plus Liquid Staining 19 Experiment Results and Study Questions 20 Instructor's Guidelines Notes to the Instructor 21 Pre-Lab Preparations 23 Batch Agarose Gel Preparation 26 Experiment Results and Analysis 27 Study Questions and Answers 28 Material Safety Data Sheets 29 EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

3 3 Experiment Components ELECTROPHORESIS SAMPLES Ready-to-Load DNA samples A DNA Standard marker B Crime scene PCR reaction C Suspect 1 PCR reaction D Suspect 2 PCR reaction E Suspect 3 PCR reaction Store entire experiment at room temperature. DNA samples do not require heating prior to gel loading. REAGENTS & SUPPLIES: Practice Gel Loading Solution UltraSpec-Agarose powder Concentrated electrophoresis buffer InstaStain Methylene Blue Methylene Blue Plus 1 ml pipet 100 ml graduated cylinder (packaging for samples) Microtipped Transfer Pipets THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. Requirements All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. Horizontal gel electrophoresis apparatus D.C. power supply Automatic micropipets with tips Balance Microwave, hot plate or burner Pipet pump 250 ml flasks or beakers Hot gloves Safety goggles and disposable laboratory gloves Small plastic trays or large weigh boats (for gel destaining) DNA visualization system (white light) Distilled or deionized water EDVOTEK - The Biotechnology Education Company EDVOTEK 24-hour FAX: (301) edvotek@aol.com

4 4 DNA Fingerprinting Background Information CRIME SCENE SUSPECT #1 Hair Skin Cells Biological Stain Crime Scene Suspect #1 Blood Draw Treat to release DNA Perform PCR to amplify specific polymorphic regions Suspect #2 Suspect #2 matches Crime Scene SUSPECT #2 Blood Draw Deoxyribonucleic acid (DNA), present in the nucleus of every living cell, is the genetic material that acts as a blueprint for all of the proteins synthesized by cells. In mammals, a large fraction of the total DNA does not encode for proteins. Polymorphic DNA refers to chromosomal regions that vary widely from individual to individual. By examining several of these regions within the genomic DNA obtained from an individual, one may determine a DNA Fingerprint for that individual. DNA polymorphisms are now widely used for determining paternity/maternity, kinship, identification of human remains, and the genetic basis of various diseases. The most widely used and far-reaching application, however, has been to the field of criminal forensics. DNA from both crime victims and offenders can now be definitively matched to crime scenes, often affecting the outcome of criminal and civil trials. The beginning of DNA fingerprinting occurred in the United Kingdom in 1984, following the pioneering work of Dr. Alex Jeffreys at the University of Leicester. Analysis by Jeffreys led to the apprehension of a murderer in the first DNA fingerprinting case in September The first U.S. conviction occurred on November 6, 1987 in Orlando, FL. Since then, DNA analysis has been used in thousands of convictions. Additionally, over 70 convicted prison inmates have been exonerated from their crimes, including eight death row inmates. Figure 1: Comparison of crime scene DNA to DNA from suspects. In 1990, the Federal Bureau of Investigation (FBI) established the Combined DNA Index System (CODIS), a system which allows comparison of crime scene DNA to DNA profiles in a convicted offender and a forensic (crime scene) index. A match of crime scene DNA to a profile in the convicted offender index indicates a suspect for the crime, whereas a match of crime scene DNA to the forensic index indicates a serial offender. CODIS has now been used to solve dozens of cases where authorities had no suspect for the crime under investigation.

5 Background Information 5 DNA Fingerprinting Target Sequence = Separation of two DNA strands = Primer 1 = Primer 2 Denature 94 C Cycle 1 Anneal 2 primers 45 C Extension 72 C Cycle 2 Cycle 3 Figure 2: The Polymerase Chain Reaction

6 6 DNA Fingerprinting Background Information The first step in forensic DNA fingerprinting is the collection of blood or tissue samples from the crime scene or victim (Figure 1). A blood sample, often present as a stain, is treated with detergent to rupture cell membranes and obtain DNA for further analysis. The early method, called restriction fragment length polymorphism (RFLP) analysis, involves digesting the DNA with restriction enzymes, separation on an agarose gel, transferring the DNA to a membrane, and hybridizing the DNA on the membrane with probes to polymorphic regions. This method requires relatively large amounts of DNA and takes several weeks to complete. More recently, the polymerase chain reaction (PCR) has been used in forensics to analyze DNA (Figure 2). This technique requires about 500- fold DNA than RFLP analysis and is less time-consuming. PCR amplification (Figure 2) uses an enzyme known as Taq polymerase. This enzyme, originally purified from a bacterium that inhabits hot springs, is stable at very high (near boiling) temperatures. Also included in the PCR reaction mixture are two synthetic oligonucleotides known as primers and the extracted DNA. The region of DNA to be amplified is known as the target. In the first step of the PCR reaction, the template complementary DNA strands are separated (denatured) from each other at 94 o C, while the Taq polymerase remains stable. In the second step, known as annealing, the sample is cooled to an intermediate temperature, usually 40 o -65 o C, to allow hybridization of the two primers, one to each of the two strands of the template DNA. In the third step, known as extension, the temperature is raised to 72 o C and the Taq polymerase adds nucleotides to the primers to complete the synthesis of the new complementary strands. These three steps - denaturation, annealing, and extension - constitute one PCR cycle. This process is typically repeated for cycles, amplifying the target sequence within DNA exponentially (Figure 2). PCR is performed in a thermal cycler, an instrument that is programmed to rapidly heat, cool and maintain samples at designated temperatures for varying amounts of time. In forensics, PCR is used to amplify and examine highly variable (polymorphic) DNA regions. These are regions that vary in length from individual to individual and fall into two categories: 1) variable number of tandem repeats (VNTR) and 2) STR (short tandem repeats). A VNTR is a region that is variably composed of a base pair sequence, typically repeated times. An STR is similar to a VNTR except that the repeated unit is only 2-4 nucleotides in length. By examining several different VNTRs or STRs from the same individual, investigators obtain a unique DNA profile for that individual which is unlike that of any other person (except for identical twins).

7 Experiment Procedures 7 Experiment Overview Remove end blocks and comb, then submerge gel under buffer in the electrophoresis chamber Prepare agarose gel in casting tray EXPERIMENT BRIEF DESCRIPTION This experiment demonstrates a PCR reaction that has been performed on hair obtained from a murder scene. Students will separate this DNA sample by agarose gel electrophoresis and compare the preamplified DNA from two possible suspects to determine if either suspect was present at the crime scene. Load each sample in consecutive wells A B C D E EXPERIMENT OBJECTIVE: The objective of this experiment is to develop a basic understanding of DNA Fingerprinting. Students will analyze PCR reactions obtained from different suspects and compare them to a crime scene sample. Attach safety cover, connect leads to power source and conduct electrophoresis Analysis on white light source after destaining After electrophoresis, transfer gel for staining InstaStain M etblue InstaStain Methylene Blue Gel Requirements Recommended gel tray size: 7 x 7 cm or 7 x 15 cm Number of sample wells required: 5 Placement of well-former template: first set of notches Agarose gel concentration required: 0.8%

8 8 Agarose Gel Preparation LABORATORY SAFETY Experiment Procedures Wear gloves and safety goggles 1. Gloves and goggles should be worn routinely as good laboratory practice. 2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents. 3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS. 4. Exercise caution when using any electrical equipment in the laboratory. 5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory. PREPARING THE GEL BED 1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape. A. Using Rubber dams: Place a rubber dam on each end of the bed. Make sure the rubber dam fits firmly in contact with the sides and bottom of the bed. B. Taping with labeling or masking tape: With 3/4 inch wide tape, extend the tape over the sides and bottom edge of the bed. Fold the extended edges of the tape back onto the sides and bottom. Press contact points firmly to form a good seal. 2. Place a well-former template (comb) in the first set of notches at the end of the bed. Make sure the comb sits firmly and evenly across the bed.

9 Experiment Procedures 9 Agarose Gel Preparation CASTING AGAROSE GELS 3. Use a 250 ml flask to prepare the gel solution. Add the following components to the flask as specified for your experiment (refer to Table A). Buffer concentrate Distilled water Agarose powder Table A Individual 0.8% UltraSpec-Agarose Gel DNA Staining with InstaStain MetBlue Size of EDVOTEK Casting Tray (cm) Amt of Agarose (g) Concentrated Distilled + Buffer (50x) + Water = (ml) (ml) Total Volume (ml) 7 x x Swirl the mixture to disperse clumps of agarose powder. 5. With a marking pen, indicate the level of the solution volume on the outside of the flask. 6. Heat the mixture to dissolve the agarose powder. The final solution should appear clear (like water) without any undissolved particles. At high altitudes, it is recommended to use a microwave oven to reach boiling temperatures. A. Microwave method: Cover the flask with plastic wrap to minimize evaporation. Heat the mixture on High for 1 minute. Swirl the mixture and heat on High in bursts of 25 seconds until all the agarose is completely dissolved. B. Hot plate method: Cover the flask with aluminum foil to prevent excess evaporation. Heat the mixture to boiling over a burner with occasional swirling. Boil until all the agarose is completely dissolved. Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved.

10 10 Agarose Gel Preparation Experiment Procedures 7. Cool the agarose solution to 55 C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step 5. After the gel is cooled to 55 C: Cool the agarose to 55 C DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED. Hot agarose solution may irreversibly warp the bed. If you are using rubber dams, go to step 9. If you are using tape, continue with step Seal the interface of the gel bed and tape to prevent the agarose solution from leaking. Use a transfer pipet to deposit a small amount of cooled agarose to both inside ends of the bed. Wait approximately 1 minute for the agarose to solidify. 9. Pour the cooled agarose solution into the bed. Make sure the bed is on a level surface. 10. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes.

11 Experiment Procedures 11 Agarose Gel Preparation PREPARING THE GEL FOR ELECTROPHORESIS 11. After the gel is completely solidified, carefully and slowly remove the rubber dams or tape from the gel bed. Be especially careful not to damage or tear the gel wells when removing the rubber dams. A thin plastic knife, spatula or pipet tip can be inserted between the gel and the dams to break possible surface tension. 12. Remove the comb by slowly pulling straight up. Do this carefully and evenly to prevent tearing the sample wells. 13. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform. 14. Fill the electrophoresis apparatus chamber with the required volume of diluted buffer for the specific unit you are using (see guidelines in Table B). For DNA analysis, the same EDVOTEK 50x Electrophoresis Buffer is used for preparing both the agarose gel buffer and the chamber buffer. The formula for diluting EDVOTEK (50x) concentrated buffer is 1 volume of buffer concentrate to every 49 volumes of distilled or deionized water. The electrophoresis (chamber) buffer recommended is Trisacetate-EDTA (20 mm Tris, 6 mm sodium acetate, 1 mm disodium ethylenediamine tetraacetic acid) ph 7.8. Prepare the buffer as required for your electrophoresis apparatus. Table B EDVOTEK Model # M6+ M12 M36 (blue) M36 (clear) Dilution of Electrophoresis (Chamber) Buffer Concentrated Buffer (50x) (ml) Distilled Water (ml) = Total Volume (ml) Make sure the gel is completely covered with buffer. 16. Proceed to loading the samples and conducting electrophoresis.

12 12 Sample Delivery (Gel Loading) PRACTICE GEL LOADING Experiment Procedures Accurate sample delivery technique ensures the best possible gel results. Pipeting mistakes can cause the sample to become diluted with buffer, or cause damage to the wells with the pipet tip while loading the gel. If you are unfamiliar with loading samples in agarose gels, it is recommended that you practice sample delivery techniques before conducting the actual experiment. EDVOTEK electrophoresis experiments contain a tube of practice gel loading solution for this purpose. Casting of a separate practice gel is highly recommended. One suggested activity is outlined below: 1. Cast a gel with the maximum number of wells possible. 2. After the gel solidifies, place it under buffer in an electrophoresis apparatus chamber. Alternatively, your teacher may have cut the gel into sections between the rows of wells. Place a gel section with wells into a small, shallow tray and submerge it under buffer or water. Note: The agarose gel is sometimes called a "submarine gel" because it is submerged under buffer for sample loading and electrophoretic separation. 3. Practice delivering the practice gel loading solution to the sample wells. Take care not to damage or puncture the wells with the pipet tip. If you are using transfer pipets, gently squeeze the pipet stem, instead of the bulb to help control the delivery of small sample volumes. For electrophoresis of DNA to be stained with InstaStain Methylene Blue, load the sample well with microliters of sample. If using transfer pipets for sample delivery, load each sample well until it is full. 4. If you need more practice, remove the practice gel loading solution by squirting buffer into the wells with a transfer pipet. 5. Replace the practice gel with a fresh gel for the actual experiment. Note: If practice gel loading is performed in the electrophoresis chamber, the practice gel loading solution will become diluted in the buffer in the apparatus. A small amount of practice gel loading solution (filling up to 12 wells) will not interfere with the experiment, so it is not necessary to prepare fresh buffer.

13 Experiment Procedures 13 Conducting Agarose Gel Electrophoresis ELECTROPHORESIS SAMPLES Samples in EDVOTEK Series 100 and Sci-On series electrophoresis experiments are packaged in one of two different formats: Pre-aliquoted QuickStrip connected tubes (new format) or Individual 1.5 ml or 0.5 ml microtest tubes Pre-aliquoted QuickStrip connected tubes Each set of QuickStrip connected tubes contains pre-aliquoted ready-to-load samples for one gel. A protective overlay covers the strip of QuickStrip sample tubes. Check the sample volume. Sometimes a small amount of sample will cling to the walls of the tubes. Make sure the entire volume of sample is at the the bottom of the tubes before starting to load the gel. A B EDVOTEK QuickStrips C D E F Tap the overlay cover on top of the strip, or tap the entire QuickStrip on the table to make samples fall to the bottom of the tubes QuickStrips patent pending Individual 1.5 ml or 0.5 ml microtest tubes Your instructor may have aliquoted samples into a set of tubes for each lab group. Alternatively, you may be required to withdraw the appropriate amount of sample from the experiment stock tubes. Check the sample volume. Sometimes a small amount of sample will cling to the walls of the tubes. Make sure the entire volume of sample is at the the bottom of the tubes before starting to load the gel. Briefly centrifuge the sample tubes, or tap each tube on the tabletop to get all the sample to the bottom of the tube.

14 14 Conducting Agarose Gel Electrophoresis Experiment Procedures QuickStrip Samples Successful Pipetting with Micropipets 1. Do not disturb the samples in the QuickStrip. Gently tap the QuickStrip tubes on the lab bench to ensure that samples are at the bottom of the tubes. * If a sample becomes displaced while inserting the pipet tip in the tube, gently tap the QuickStrip on the lab bench to concentrate the sample to the bottom of the tube. With the pipet plunger depressed to the first stop, re-insert the tip into the sample and raise the micropipet plunger to withdraw the sample. Delivering QuickStrip Samples with Transfer Pipets: If using disposable transfer pipets for sample delivery, pierce the protective overlay with a paper clip before inserting the transfer pipet to withdraw the sample. 2. Stabilize the QuickStrip by firmly anchoring it on the lab bench. 3. Gently pierce the printed protective overlay with the pipet tip attached to a micropipet. Depress the micropipet plunger to the first stop before the tip is placed in contact with the sample. 4. With the pipet plunger depressed to the first stop, insert the tip into the sample. 5. Raise the plunger of the micropipet to withdraw the sample. 6. Load the sample into the appropriate well of the gel. Discard the tip. 7. Repeat steps 3-6 for each sample. Reminder: During electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode. Before loading the samples, make - + Black Red sure the gel is Sample wells properly oriented in the apparatus chamber. LOAD THE SAMPLES For either QuickStrip or individual microtest tube format, samples should be loaded into the wells of the gel in consecutive order. Load the DNA samples in tubes A - E into the wells in consecutive order. The amount of sample that should be loaded is µl. A B C D E DNA Standard marker Crime scene PCR reaction Suspect 1 PCR reaction Suspect 2 PCR reaction Suspect 3 PCR reaction

15 Experiment Procedures 15 Conducting Agarose Gel Electrophoresis RUNNING THE GEL 1. After the DNA samples are loaded, carefully snap the cover down onto the electrode terminals. Make sure that the negative and positive color-coded indicators on the cover and apparatus chamber are properly oriented. 2. Insert the plug of the black wire into the black input of the power source (negative input). Insert the plug of the red wire into the red input of the power source (positive input). 3. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor. General guidelines are presented in Table C. Time and Voltage Electrophoresis of DNA Table C 4. Check to see that current is flowing properly - you should see bubbles forming on the two platinum electrodes. Volts Recommended Time Minimum Maximum 30 min 40 min 60 min 40 min 75 min 100 min 5. After the electrophoresis is completed, turn off the power, unplug the power source, disconnect the leads and remove the cover. 6. Remove the gel from the bed for staining with InstaStain Methylene Blue.

16 16 Staining & Visualization of DNA: InstaStain Methylene Blue Experiment Procedures Revised Instructions InstaStain Methylene Blue Wear gloves and safety goggles EDVOTEK electrophoresis experiments feature a state-of-the-art, proprietary stain for DNA gels called InstaStain. This experiment features InstaStain Methylene Blue, sometimes abbreviated as "InstaStain MetBlue", which saves time and reduces liquid waste. Methylene blue stain is released when the card is placed in contact with the moist gel. InstaStain Ethidium Bromide is also available from EDVOTEK. 1 3 Place gel on a flat surface covered with plastic wrap DNA InstaStain Patents Pending Press firmly. STAINING OF DNA 1. After electrophoresis, place the agarose gel on a flat surface covered with plastic wrap. 2. Wearing gloves, place the blue dye side of the InstaStain Methylene Blue card on the gel. 3. Firmly run your fingers several times over the entire surface of the InstaStain card to establish good contact between the InstaStain card and the gel. 4. To ensure continuous contact between the gel and the InstaStain card, place a gel casting tray and weight, such as a small empty beaker, on top of the InstaStain card. 2 4 InstaStain InstaStain Patents Pending Place the InstaStain card on the gel Patents Pending Place a small weight for approx. 5 minutes. Visit our web site for animated demontrations of InstaStain. 5. Allow the InstaStain Methylene Blue to sit on the gel for 5 to 10 minutes. 6. After staining, remove the InstaStain card. If the color of the gel appears very light, wet the gel surface with buffer or distilled water and place the InstaStain card back on the gel for an additional 5 minutes. InstaStain is a registered trademark of EDVOTEK, Inc. Patents Pending.

17 Experiment Procedures 17 Staining & Visualization of DNA: InstaStain Methylene Blue DESTAINING AND VISUALIZATION OF DNA 7. Transfer the gel to a large weigh boat or small plastic container. 8. Destain with distilled water.* 5 Add approximately 100 ml of distilled water to cover the gel. Repeat destaining by changing the distilled water as needed. 6 Transfer to a small tray for destaining. * Destaining Notes Warmed distilled water at 37 C will accelerate destaining. Destaining will take longer with room temperature water. DO NOT EXCEED 37 C! Warmer temperatures will soften the gel and may cause it to break. The volume of distilled water for destaining depends upon the size of the tray. Use the smallest tray available that will accommodate the gel. The gel should be completely submerged during destaining. Do not exceed 3 changes of water for destaining. Excessive destaining will cause the bands to be very light. Destain with 37 C distilled water The larger DNA bands will initially be visible as dark blue bands against a lighter blue background. When the gel is completely destained, the larger DNA bands will become sharper and the smaller bands will be visible. With additional destaining, the entire background will become uniformly light blue. 9. Carefully remove the gel from the destain solution and examine the gel on a Visible Light Gel Visualization System. To optimize visibility, use the amber filter provided with EDVOTEK equipment. 10. If the gel is too light and bands are difficult to see, repeat the staining and destaining procedures. InstaStain is a registered trademark of EDVOTEK, Inc. Patents Pending

18 18 Staining & Visualization of DNA: InstaStain Methylene Blue Experiment Procedures NEW Examples of small trays include large weigh boats, or small plastic food containers EASY ONE-STEP STAINING AND DESTAINING OVERNIGHT WITH INSTASTAIN METBLUE Agarose gels can be stained overnight with InstaStain Methylene Blue cards in one easy step. This one-step method is an excellent alternative if time does not permit staining during a regular class session. Instructions for staining a 7 x 7 cm gel after electrophoresis follow. 1. Remove the 7 x 7 cm agarose gel from its bed and totally submerse the gel in a small, clean tray containing 75 ml of distilled or deionized water, or used electrophoresis buffer. The agarose gel should be completely covered with liquid. Do not stain gel(s) in the electrophoresis apparatus. 2. Gently float a 7 x 7 cm card of InstaStain MetBlue with the stain side (blue) facing the liquid. 3. Let the gel soak undisturbed in the liquid overnight. The gel will be stained, destained and ready for photography the next day. STORAGE AND DISPOSAL OF INSTASTAIN METHYLENE BLUE CARDS AND GELS Stained gels may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid. DO NOT FREEZE AGAROSE GELS! Used InstaStain cards and destained gels can be discarded in solid waste disposal. Destaining solutions can be disposed down the drain.

19 Experiment Procedures 19 Staining & Visualization of DNA: Methylene Blue Plus Liquid Staining LIQUID STAINING AND DESTAINING OF DNA 1. Remove each agarose gel from its bed and totally submerse up to 6 gels in a tray containing 600 ml of diluted Methylene Blue Plus stain. Do not stain gel(s) in the electrophoresis apparatus. Each group should mark their gel, such as removing a small slice, or making a small hole in a designated corner, to facilitate identification after staining and destaining. Wear gloves and safety goggles 2. Stain gel(s) for a minimum of 30 minutes, with occasional agitation. 3. Destain in 600 ml of distilled water that has been warmed to 37 C. Dilution of Methylene Blue Plus stain: Dilute the 10x stain by mixing 1 part stain with 9 parts distilled or deionized water. Completely submerse the gel(s) in 600 ml of 37 C distilled water for 15 minutes with occasional agitation. Then discard the destaining solution Change the distilled water for a second destain for another 15 minutes with occasional agitation. Bands will become visible after the second destain. You may also leave the gel(s) in destain overnight. DO NOT EXCEED 37 C! Warmer temperatures will soften the gel and may cause it to break. 4. Carefully remove the gel from the destain solution and examine on a Visible Light Gel Visualization System. To optimize visibility, use the amber filter provided with EDVOTEK equipment. 5. If the gel is too light and bands are difficult to see, repeat the staining and destaining procedures. STORAGE AND DISPOSAL METHYLENE BLUE PLUS STAIN AND GEL Gels stained with Methylene Blue Plus may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid. DO NOT FREEZE AGAROSE GELS. Stained gels which are not kept can be discarded in solid waste disposal. Methylene Blue Plus stain and destaining solutions can be disposed down the drain.

20 20 Experiment Results and Study Questions LABORATORY NOTEBOOK RECORDINGS: Experiment Procedures Address and record the following in your laboratory notebook or on a separate worksheet. Before starting the experiment: Write a hypothesis that reflects the experiment. Predict experimental outcomes. During the Experiment: Record (draw) your observations, or photograph the results. Following the Experiment: Formulate an explanation from the results. Determine what could be changed in the experiment if the experiment were repeated. Write a hypothesis that would reflect this change. STUDY QUESTIONS Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. What is polymorphic DNA? How is it used for identification purposes? 2. What is CODIS? How is it used to solve crimes? 3. What is an STR? A VNTR? Which (STR or VNTR) is predominantly now used in law enforcement? Why?

21 21 Notes to the Instructor Online Ordering now available Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at EDVOTEK ( ). NATIONAL CONTENT AND SKILL STANDARDS By performing this experiment, students will learn to load samples and run agarose gel electrophoresis. Analysis of the experiments will provide students the means to transform an abstract concept into a concrete explanation. Please visit our website for specific content and skill standards for various experiments. Instructor's Guide EDUCATIONAL RESOURCES Visit our web site for information about EDVOTEK's complete line of experiments for biotechnology and biology education. M o n EDVO-TECH EDVOTEK - ( ) Fri 9 SERVICE am - 6 E T pm Electrophoresis Hints, Help and Frequently Asked Questions EDVOTEK Ready-to-Load Electrophoresis Experiments are easy to perform and are designed for maximum success in the classroom setting. However, even the most experienced students and teachers occasionally encounter experimental problems or difficulties. The Technical Service EDVOTEK web site provides several suggestions and reminders for conducting electro- Department phoresis, as well as answers to frequently asked electrophoresis questions. Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) web: edvotek@aol.com Please have the following information: The experiment number and title Kit Lot number on box or tube The literature version number (in lower right corner) Approximate purchase date Laboratory Extensions and Supplemental Activities Laboratory extensions are easy to perform using EDVOTEK experiment kits. For example, a DNA sizing determination activitiy can be performed on any electrophoresis gel result containing DNA markers run in parallel with other DNA samples. For DNA Sizing instructions, and other laboratory extension suggestions, please refer to the EDVOTEK website, which is updated on a continous basis with educational activities and resources.

22 22 Notes to the Instructor: Instructor's Guide EDVOTEK Experiment # S-44, Micropipetting Basics, focuses exclusively on the use of micropipets. Students learn and practice pipeting techniques by preparing various dye mixtures and deliver their mixture to a special Pipet Card. APPROXIMATE TIME REQUIREMENTS 1. Gel preparation: Whether you choose to prepare the gel(s) in advance or have the students prepare their own, allow approximately minutes for this procedure. Generally, 20 minutes of this time is required for gel solidification. 2. Practice Gel Loading: If your students are unfamiliar with using micropipets and sample loading techniques, a practice activity is suggested prior to conducting the experiment. This activity can require anywhere from 10 minutes to an entire laboratory session, depending upon the skill level of your students. 3. Conducting Electrophoresis: The approximate time for electrophoresis will vary from 30 minutes to 2 hours depending upon the voltage used. Generally, the higher the voltage applied the faster the samples migrate. However, the maximum amount of voltage significantly depends upon the design of the electrophoresis apparatus and should not exceed manufacturer's recommendations. Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C. Time and Voltage Electrophoresis of DNA Table C 4. Recommended Time Staining and Destaining: Volts Minimum Maximum DNA gel staining with InstaStain Methylene Blue requires less time than traditional liquid staining methods. Staining requires approximately 5-10 minutes. DNA bands will become visible after destaining for approximately 20 minutes, and will min 40 min 60 min become sharper with additional destaining time. 40 min 75 min 100 min 5. Photography of Gels Stained with InstaStain MetBlue: Although bands will be visible after destaining per instructions, additional destaining is recommended for the best photographic results. After staining with InstaStain Methylene Blue, allow the gel to remain in destain solution for several hours to overnight. This will allow the stained gel to "equilibrate" in the destaining solution, allowing the dark blue DNA bands to fully contrast against a uniformly light blue background.

23 23 Pre-Lab Preparations PREPARING GELS FOR PRACTICE GEL LOADING If your students are not familiar with loading samples in agarose gels, it is recommended that they practice sample delivery techniques before conducting the actual experiment. EDVOTEK electrophoresis experiments contain a tube of practice gel loading solution for this purpose. Casting of a separate practice gel is highly recommended. PREPARING GELS FOR ELECTROPHORESIS There are several options for preparing agarose gels for the electrophoresis experiments: Instructor's Guide 1. Individual Gel Casting Each student lab group can be responsible for casting their own individual gel prior to conducting the experiment. 2. Batch Gel Preparation A batch of agarose gel can be prepared for sharing by the class. To save time, a larger quantity of UltraSpec-Agarose can be prepared for sharing by the class. See instructions for "Batch Gel Preparation". 3. Preparing Gels in Advance Gels may be prepared ahead and stored for later use. Solidified gels can be stored under buffer in the refrigerator for up to 2 weeks. Do not store gels at -20 C. Freezing will destroy the gels. Gels that have been removed from their trays for storage, should be "anchored" back to the tray with a few drops of hot, molten agarose before placing the gels into the apparatus for electrophoresis. This will prevent the gels from sliding around in the trays and the chambers.

24 24 Pre-Lab Preparations Instructor's Guide READY-TO-LOAD DNA SAMPLES FOR ELECTROPHORESIS No heating required before gel loading. EDVOTEK offers the widest selection of electrophoresis experiments which minimize expensive equipment requirements and save valuable time for integrating important biotechnology concepts in the teaching laboratory. Series 100 experiments feature DNA samples which are predigested with with restriction enzymes and are stable at room temperature. DNA samples are ready for immediate delivery onto agarose gels for electrophoretic separation and do not require pre-heating in a waterbath. Electrophoresis samples and reagents in EDVOTEK experiments are packaged in various formats. The samples in Series 100 and S-series electrophoresis experiments will be packaged in one of the following ways: 1) Pre-aliquoted Quickstrip connected tubes OR 2) Individual 1.5 ml (or 0.5 ml) microtest tubes FORMAT: PRE-ALIQUOTED QUICKSTRIP CONNECTED TUBES EDVOTEK DO NOT BEND A B C D E F G H CUT HERE A B C D E F G H CUT HERE A B C D E F G H CUT HERE A B C D E F G H CUT HERE A B C D E F G H CUT HERE Carefully cut between each set of tubes A B C D E F G H Convenient QuickStrip connected tubes contain pre-aliquoted ready-to-load samples. The samples are packaged in a microtiter block of tubes covered with a protective overlay. Pre-lab preparation requires only simple separation of tubes into strips for a complete set of samples for one gel. 1. Use sharp scissors to separate the block of samples into individual strips as shown in the diagram at left. Each row of samples (strip) constitutes a complete set of samples for each gel. The number of samples per set will vary depending on the experiment. Some tubes may be empty. A B C D E F 2. Cut carefully between the rows of samples. Do not cut or puncture the protective overlay directly covering the sample tubes. 3. Each group will require one strip of samples. 4. Remind students to tap the tubes before gel loading to ensure that all of the sample is at the bottom of the tube.

25 25 Pre-Lab Preparations FORMAT: INDIVIDUAL 1.5 ML MICROTEST TUBES It is recommended that samples packaged in 1.5 ml individual microtest tubes be aliquoted for each gel. Samples packaged in this format include bulk samples for EDVOTEK Series 100 electrophoresis experiments and are available in two standard quantities: the B-Series (480 µl) and the C Series (960 µl). Custom bulk quantities are also available by request. Before aliquoting, check all sample volumes for possible evaporation. The samples will become more concentrated if evaporation has occurred. Instructor's Guide If needed, tap or centrifuge the sample tubes. Then add distilled water to slightly above the following level: 2.3 cm level for the B-Series 3.3 cm level for the C-Series 4.7 cm 2.3 cm 3.3 cm Mix well by inverting and tapping the tubes several times. After checking sample volumes and determining that the samples are at their proper total volumes: B Series C Series 1. Aliquot the samples into appropriately labeled 0.5 ml or 1.5 ml microtest tubes. For gels to be stained with InstaStain Methylene Blue: µl of each sample 2. Students might have difficulty retrieving the entire aliquoted volume of sample because some of it may cling to the side walls of the tubes. Some suggestions are: Remind students to make sure all of the sample is at the bottom of the tube before gel loading. They should centrifuge the samples tubes, or tap the tubes on the tabletop. Instruct students to set their automatic micropipets to a volume that is 2 microliters less than the volume you have aliquoted.

26 26 Batch Agarose Gel Preparation Instructor's Guide To save time, the agarose gel solution can be prepared in a batch for sharing by the class. Any unused prepared agarose can be saved and remelted for gel casting at a later time. For a batch (375 ml) preparation of 0.8% agarose gel: 1. Use a 500 ml flask to prepare the diluted gel buffer. Add 7.5 ml of buffer concentrate Add ml of distilled water. 2. Pour 3.0 grams of UltraSpec-Agarose into the prepared buffer. Swirl to disperse clumps. Note: The UltraSpec-Agarose kit component is often labeled with the amount it contains. In many cases, the entire contents of the bottle is 3.0 grams. Please read the label carefully. If the amount of agarose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount. 3. With a marking pen, indicate the level of solution volume on the outside of the flask. 4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution. 5. Cool the agarose solution to 55 C with swirling to promote even dissipation of heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume (375 ml) as marked on the flask in step C Table D Amt of Agarose (g) Batch Preparation of 0.8% UltraSpec-Agarose Concentrated Distilled + Buffer (50x) + Water = (ml) (ml) Total Volume (ml) Dispense the required volume of cooled agarose solution for casting the gels. The volume required is dependent upon the size of the gel bed (refer to Table A for individual gel casting guidelines). 7. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. Then proceed with preparing the gel for electrophoresis.

27 27 Experiment Results and Analysis To view a color photo of the gel result, refer to the EDVOTEK Catalog listing for this experiment, or visit the EDVOTEK web site at Photo of gel result Lane Tube 1 A DNA Standard marker 2 B Crime scene PCR reaction 3 C Suspect 1 PCR reaction 4 D Suspect 2 PCR reaction 5 E Suspect 3 PCR reaction In the idealized schematic, the relative positions of DNA fragments are shown but are not depicted to scale. Instructor's Guide The DNA standards in Lane 1 make it possible to measure the DNA bands obtained from the PCR reactions. The results of this analysis indicates an identical pattern in Lanes 2 and 4. This is strong evidence that the crime scene DNA and Suspect 2 match. In criminal investiations, several known variable regions in DNA are analyzed to match crime scene and suspect DNAs. Laboratory notebooks It is highly recommended that students maintain a laboratory notebook to formulate hypotheses and to record experimental procedures and results. EDVOTEK offers two student laboratory notebooks. Call EDVOTEK ( ) for classroom quantity pricing information. Cat. # 1401 Cat. # 1402 Laboratory Notebook, 50 pages Laboratory Notebook, 100 pages

28 28 Study Questions and Answers 1. What is polymorphic DNA? How is it used for identification purposes? Instructor's Guide Polymorphic DNA refers to chromosomal regions that vary widely from person to person. This variation is usually in the length of a specific DNA region. By analyzing a number of these regions, one may obtain a DNA fingerprint of a person that will not match the DNA fingerprint of any other individual. DNA fingerprinting is used for identification of missing persons, human remains, and matching criminal suspects to crime scenes. 2. What is CODIS? How is it used to solve crimes? CODIS is an acronym for the COmbined DNA Index System, a computer-based database containing DNA fingerprints. In the convicted offender database, DNA profiles of convicted felons are maintained. In the forensic database, DNA fingerprints from crime scenes are maintained. 3. What is an STR? A VNTR? Which (STR or VNTR) is predominantly now used in law enforcement? Why? An STR is an acronym for a short tandem repeat, a DNA sequence of 2-4 base pairs that is repeated variably from person to person. VNTRs, or variable number of tandem repeats, have longer repeat units of base pairs. STRs, are now preferred to VNTRs as the length of their amplified products requires less template DNA to be amplified.

29 EDVOTEK Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Stable Incompatibility available X Conditions to Avoid IDENTITY (As Used on Label and List) Agarose Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Other Limits Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. CAS # Section III - Physical/Chemical Characteristics Boiling Point For 1% solution 194 F Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl Acetate = 1) Solubility in Water Insoluble - cold Appearance and Odor White powder, no odor Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL N.D. UEL N.D. Extinguishing Media Water spray, dry chemical, carbon dioxide, halon or standard foam Special Fire Fighting Procedures Possible fire hazard when exposed to heat or flame 07/01/03 Hazardous Decomposition or Byproducts Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Inhalation: available Ingestion: Large amounts may cause diarrhea Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure available Medical Conditions Generally Aggravated by Exposure available Emergency First Aid Procedures Treat symptomatically and supportively Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method Precautions to be Taken in Handling and Storing Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Special Protective Gloves Yes Sweep up and place in suitable container for disposal Normal solid waste disposal Chemical cartridge respirator with full facepiece. Mechanical (General)Gen. dilution ventilationother Eye Protection Splash proof goggles Unusual Fire and Explosion Hazards Other Protective Clothing or Equipment Work/Hygienic Practices Impervious clothing to prevent skin contact EDVOTEK Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X Incompatibility Strong oxidizing agents IDENTITY (As Used on Label and List) 50x Electrophoresis Buffer Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. 07/01/03 Other Limits Recommended % (Optional) Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Melting Point Evaporation Rate (Butyl Acetate = 1) Solubility in Water Appearance and Odor Appreciable, (greater than 10%) Clear, liquid, slight vinegar odor Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL UEL N.D. N.D. Extinguishing Media Use extinguishing media appropriate for surrounding fire. Special Fire Fighting Procedures Wear protective equipment and SCBA with full facepiece operated in positive pressure mode. Unusual Fire and Explosion Hazards identified Hazardous Decomposition or Byproducts Carbon monoxide, Carbon dioxide Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Carcinogenicity: identified NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Irritation to upper respiratory tract, skin, eyes Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Ingestion: If conscious, give large amounts of water Eyes: Flush with water Inhalation: Move to fresh air Skin: Wash with soap and water Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Wear suitable protective clothing. Mop up spill and rinse with water, or collect in absorptive material and dispose of the absorptive material. Waste Disposal Method Dispose in accordance with all applicable federal, state, and local enviromental regulations. Precautions to be Taken in Handling and Storing Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Yes Special Mechanical (General) Yes Other Protective Gloves Yes Eye Protection Other Protective Clothing or Equipment Work/Hygienic Practices Avoid eye and skin contact. Safety goggles

30 EDVOTEK Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Stable Incompatibility X Conditions to Avoid IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must Practice Gel Loading Solution be marked to indicate that. Section I Manufacturer's Name Emergency Telephone Number (301) EDVOTEK, Inc. Telephone Number for information Address (Number, Street, City, State, Zip Code) (301) Rothgeb Drive Rockville, MD Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl Acetate = 1) Solubility in Water Soluble Appearance and Odor Blue liquid, no odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits Extinguishing Media Dry chemical, carbon dioxide, water spray or foam Unusual Fire and Explosion Hazards 07/01/03 Other Limits Recommended This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. LEL % (Optional) UEL Special Fire Fighting Procedures Use agents suitable for type of surrounding fire. Keep upwind, avoid breathing hazardous sulfur oxides and bromides. Wear SCBA. Unknown Hazardous Decomposition or Byproducts Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Acute eye contact: May cause irritation. available for other routes. Carcinogenicity: NTP? IARC Monographs? available OSHA Regulation? Signs and Symptoms of Exposure May cause skin or eye irritation Medical Conditions Generally Aggravated by Exposure reported Emergency First Aid Procedures Treat symptomatically and supportively. Rinse contacted area with copious amounts of water. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Wear eye and skin protection and mop spill area. Rinse with water. Waste Disposal Method Observe all federal, state, and local regulations. Precautions to be Taken in Handling and Storing Avoid eye and skin contact. Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Sulfur oxides, and bromides Ventilation Local Exhaust Yes Special Mechanical (General) Yes Other Protective Gloves Yes Eye Protection Other Protective Clothing or Equipment required Work/Hygienic Practices Avoid eye and skin contact Splash proof goggles EDVOTEK IDENTITY (As Used on Label and List) InstaStain Methylene Blue Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Other Limits Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended Methylene Blue 3.7 Bis (Dimethylamino) Phenothiazin 5 IUM Chloride available CAS # Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl Acetate = 1) Solubility in Water Soluble - cold Appearance and Odor Chemical bound to paper, no odor Unusual Fire and Explosion Hazards 07/01/03 % (Optional) Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL UEL available Extinguishing Media Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam Special Fire Fighting Procedures Self contained breathing apparatus and protective clothing to prevent contact with skin and eyes Emits toxid fumes under fire conditions Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X Incompatibility Strong oxidizing agents Hazardous Decomposition or Byproducts Toxic fumes of Carbon monoxide, Carbon dioxide, nitrogen oxides, sulfur oxides, hydrogen, chloride gas Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Yes Yes Ingestion? Yes Health Hazards (Acute and Chronic) Skin: May cause skin irritation Eyes: May cause eye irritation Inhalation: Cyanosis Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Meets criteria for proposed OSHA medical records rule PEREAC Signs and Symptoms of Exposure available Medical Conditions Generally Aggravated by Exposure available Emergency First Aid Procedures Treat symptomatically Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Ventilate area and wash spill site Waste Disposal Method Mix material with a combustible solvent and burn in chemical incinerator equipped with afterburner and scrubber. Check local and state regulations. Precautions to be Taken in Handling and Storing Keep tightly closed. Store in cool, dry place Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) MIOSH/OSHA approved, SCBA Ventilation Local Exhaust Special Mechanical (General) Required Other Protective Gloves Rubber Eye Protection Chem. safety goggles Other Protective Clothing or Equipment Rubber boots Work/Hygienic Practices