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EDVO-Kit #

114
DNA Paternity Testing Simulation
Storage: See Page 3 for specific storage instructions

ExPErImENT OBjECTIVE:
The objective of this experiment module is to introduce students to the use of DNA Fingerprinting in a hypothetical paternity determination.

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EDVO-Kit #

114 114

DNA Paternity Testing Simulation

Table of Contents
Page Experiment Components Experiment Requirements Background Information Experiment Procedures Experiment Overview and General Instructions Electrophoresis Gel Preparation Conducting Electrophoresis Staining and Visualization of DNA Method 1: One-Step Staining and Destaining with InstaStain® Methylene Blue Method 2: Staining with InstaStain® Methylene Blue Method 3: Liquid Staining with Methylene Blue Plus™ Study Questions Instructor's Guidelines Notes to the Instructor Pre-Lab Preparations Quantity Preparations for Agarose Gel Electrophoresis Experiment Results and Analysis Study Questions and Answers 22 23 25 26 27 27 30 32 33 34 35 16 20 11 3 3 5

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.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None

Material Safety Data Sheets

of the experiment components are derived from human sources.
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

EVT 007206AM EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

DNA Paternity Testing Simulation

EDVO-Kit # 

114

Experiment Components
DNA samples are stable at room temperature. However, if the experiment will not be conducted within one month of receipt, it is recommended that the DNA samples be stored in the refrigerator.

rEADy-TO-LOAD™ DNA SAmPLES FOr ELECTrOPhOrESIS
A B C D E Standard DNA Fragments Mother DNA cut with Enzyme Child DNA cut with Enzyme Father 1 DNA cut with Enzyme Father 2 DNA cut with Enzyme

Store entire experiment at room temperature. DNA samples do not require heating prior to gel loading.

rEAgENTS & SuPPLIES
• • • • • • • • UltraSpec-Agarose™ powder Concentrated electrophoresis buffer InstaStain® Methylene Blue Methylene Blue Plus™ Practice Gel Loading Solution 1 ml pipet 100 ml graduated cylinder (packaging for samples) Microtipped Transfer Pipets

requirements
• • • • • • • • • • • • 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

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com email: edvotek@aol.edvotek.edvotek.4 EDVO-Kit # 114 114 DNA Paternity Testing Simulation Online Ordering now available E O DV -TE C H S E RV I C E Technical Service Department Mon .com ET FAX: (301) 340-0582 Web: www.The Biotechnology Education Company® • 1-800-EDVOTEK • www.Fri 9 am Please have the following information ready: • Experiment number and title • Kit lot number on box or tube • Literature version number (in lower right corner) • Approximate purchase date EVT 007206AM EDVOTEK . 1-800-EDVOTEK (1-800-338-6835) Mo pm -6 n .Fri 9:00 am to 6:00 pm ET Visit our web site for information about EDVOTEK’s complete line of “hands-on” experiments for biotechnology and biology education.com .

and possible fathers are analyzed. Inc. The distinguishing feature of restriction enzymes is that they only cut at very specific sequences of bases called recognition sites.DNA Paternity Testing Simulation EDVO-Kit # 5 114 DNA Paternity Testing DNA fingerprinting (also called DNA typing) allows for the identification of the source of DNA samples. Copyright © 1994. Over 3. samples obtained from the mother. Restriction enzymes are endonucleases which catalyze the cleavage of the phosphate bonds within both strands of DNA. incest. citizenship of children to the United States and matching of children with parents who were mismatched at birth due 1 2 3 4 to hospital errors.000 restriction enzymes have been discovered and catalogued. Prior to the advent of the Polymerase Chain Reaction (PCR).1995. the DNA bands present in the child's fingerprint must be found in either the father's or mother's fingerprint. This document. A child's DNA is a composite of its parent DNAs. Because of allelic differences. Paternity determination based on DNA analysis (genetic DNA fingerprinting) has become an important procedure for matching children with biological fathers and mothers. in conjunction with use of accompanying reagents. immigration. For paternity DNA fingerprinting. Therefore. 2000. This is done by using the first letter of the genus followed by the first two letters of the species.1998. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.1997. such as blood typing. DNA fingerprinting can provide positive identification with great accuracy. In contrast to the more conventional methodologies. They require Mg+2 for activity and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the point of cleavage. Examples of recent court cases that have utilized this procedure have included rape. The type of strain or substrain sometimes follows the species designation in the name. This type of testing is also used during unrest as in nations in civil war where children are often separated from parents and subsequently reunited. is permitted for classroom/laboratory use only. Restriction enzymes are named according to the organism from which they are isolated. The method has become very important to provide evidence in paternity and criminal cases. or any part. Duplication of this document. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.edvotek. which can only exclude a suspect. DNA fingerprinting involved the electrophoretic analysis of DNA fragment sizes generated by restriction enzymes followed by Southern Blot Analysis. Background Information Decreasing Fragment Size Lane 1 Lane 2 Lane 3 Lane 4 Mother Child Father Unrelated Figure 1: The child's (lane 2) DNA pattern contains DNA from the mother (lane 1) and the biological father (lane 3). Only certain strains or substrains of a particular species may be a producer of restriction enzymes. 2006. Inc.com . Restriction enzymes are produced by many different species of bacteria (including blue-green algae). Bands in the child's DNA fingerprint that are not present in the mother's must be contributed by the father. comparison of DNA fragmentation patterns obtained from the mother and child will give a partial match. the child.. 2004. EDVOTEK.

In DNA paternity and fingerprinting laboratories. i. Other differences in base sequences between individuals can occur because of mutations and deletions.. Examples of sixbase cutting enzymes include Bam HI and Pst I. 2006.1997. recognition sites are Escherichia coli. It follows that alleles have differences in their base sequences which consequently creates differences in the distribution and frequencies of restriction enzyme recognition sites. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. strain RY13 frequently symmetrical.edvotek. There are several reasons for this fact. and which represent a composite of the parental genes. The two copies of a gene (which can be alleles) at a given chromosomal locus. Chromosomes occur in matching pairs. both DNA strands in the site have the same base sequence when read 5' to 3'.. Copyright © 1994.1995. which are 4-base and 5-base cutting enzymes respectively. in conjunction with use of accompanying reagents. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. one of maternal and the other of paternal origin. Inc. Inc. is permitted for classroom/laboratory use only. A large number of alleles exist in the population. Alleles are alternate forms of a gene. With some exceptions. No two individuals have exactly the same pattern of restriction enzyme recognition sites. the commonly used restriction enzymes are Hae III (GG'CC) and Hinf I (G'ANTC). The recognition sites for these restriction enzymes are: Bam HI ↓ 5'-GGATCC-3' 3'-CCTAGG-5' ↑ Pst I ↓ 5'-CTGCAG-3' 3'-GACGTC-5' ↑ The size of the DNA fragments generated by restriction enzyme cleavage depends on the distance between the recognition sites. Haemophilus influenzae Rf Such sequences are called palindromes. Background Information Bam HI Hae III Eco RI Hinf I Figure 2 Restriction enzymes recognize specific double stranded sequences in DNA. or any part.e. EDVOTEK. a Roman numeral is always used to designate one out of possibly several different restriction enzymes produced by the same organism or by different substrains of the same strain. 2000. the DNAs from a hypothetical paternity case are cut by a restriction enzyme. Such changes can also create or eliminate a recognition site. The cleavage positions are indicated by arrows. which is a six-base cutting enzyme. Duplication of this document. Cleavage occurs within or near Haemophilus aegyptius the site. constitutes the unique genotype for an offspring. Most recognition sites are 4 to 8 base pairs in length. This document. It is estimated that about 25% of all human genes occur in multiple alleles which are called polymorphisms.com . 2004. In this experiment. It is these sites in DNA that are substrates for restriction enzymes. EDVO-Kit # 114 DNA Paternity Testing DNA Paternity Testing Simulation Restriction Enzyme Organism Bacillus amyloliquefaciens Finally. Alleles result in alternative expressions of genetic traits which can be dominant or recessive and are inherited in a Mendelian pattern just as genes.1998.

Asn .. Inc. In Southern blot analysis. = repetitious sequence array (variable length) = recognition site denaturation of the DNA fragments... there are several types of these short. 2006. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. Individual variations in the distances between recognition Protein COOH sites in chromosomal DNA are often caused by intervening repetitive base 3' Codon changed sequences. The length of these arrays (the amount of repeated sets) varies between individuals at different chromosomal loci. Eco RI site 5' H 2N 5' ACG AAT TCC Thr . RFLPs are a manifestation of the unique molecular genetic profile. As shown in Figure 4..Ser Figure 3 ( Restriction Fragments Figure 4 Duplication of this document. transfer of DNA to a membrane. 2004. is permitted for classroom/laboratory use only.Ser * ACG AAC TCC Background Information H 2N Thr . Ten to fifteen percent of mammalian DNA consists of sets of repeated.1998. DNA probes are used to detect the length differences between these repetitive sequences. DNA probes are short fragments of Allele 1 Allele 2 single stranded DNA that are isotopically or non) ) ( isotopically labeled. and exposure to probes to detect DNA Fingerprints.1997. These sequences can occur between genes or are adjacent to them. 2000. smaller Southern blot analysis requires electrophoresis. in conjunction with use of accompanying reagents. Inc. DNA probes will complement larger and hybridize (attach) to single stranded DNA. in a population. RFLPs. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. Variations in the length of these fragments between different individuals.Asn .DNA Paternity Testing Simulation EDVO-Kit #  114 DNA Paternity Testing The example in Figure 3 shows how a silent mutation can eliminate a recognition site but leave a protein product 3' Coding DNA unchanged. TGTTTA | TGTTTA | TGTTTA | .com . Repetitious sequences by mutation constitute a large fraction of the mamProtein COOH malian genome and have no known genetic function. are known as restriction fragment length polymorphisms. Copyright © 1994.. or “fingerprint”. the length of the repeat will determine the size of the restriction enzyme fragment generated.. Several hundred RFLPs have been mapped on all 23 chromosomes. This document..1995.. repetitive sequences that have been cloned and purified.. or any part. of an individual’s DNA. They are also found within introns.edvotek. short sequences of bases that are tandemly arranged in arrays.variable number When these arrays are flanked by recognition sites. EDVOTEK.

one to each of the two strands of the template DNA. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. an instrument that is programmed to rapidly heat. Several SLPs are available and are used less frequently since more than one person can exhibit the same exact pattern for a specific SLP.denaturation. The region of DNA to be amplified is known as the “target”. the temperature is raised to 72°C and the Taq polymerase adds nucleotides to the primers to synthesize the new complementary strands. in conjunction with use of accompanying reagents. is permitted for classroom/laboratory use only. PCR is used to amplify and examine highly variable (polymorphic) DNA regions. EDVOTEK. amplifying the target sequence within DNA exponentially (Figure 5).8 EDVO-Kit # 114 DNA Paternity Testing DNA Paternity Testing Simulation Background Information There are two types of probes commonly used for genetic identification. while the Taq polymerase remains stable. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. originally was purified from a bacterium that inhabits hot springs and is stable at very high (near boiling) temperatures.. the template complementary DNA strands are separated (denatured) from each other at 94°C. Copyright © 1994.edvotek. Multiple-Locus probes (MLPs) detect multiple repetitive DNA segments located on many chromosomes yielding 20-30 bands. In forensics. 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). If the segments on the chromosome pairs are the same.1997. In the second step.1995. Inc. The PCR products are separated by agarose gel electrophoresis and DNA fingerprints are analyzed. Currently. These three steps . if they are different. This process is typically repeated for 20-40 cycles. Because of the multi-band patterns. known as extension. it is calculated that two unrelated individuals having the identical DNA pattern detected by MLPs as an average is 1 in 30 billion. 2000. then there will be one band. An STR is similar to a VNTR except that the repeated unit is Duplication of this document. it will appear as two bands. the polymerase chain reaction (PCR) is routinely used in forensics to analyze DNA (Figure 4). 2006. to allow hybridization of the two primers. cool and maintain samples at designated temperatures for varying amounts of time. annealing. or any part.com . This technique requires about 500-fold less DNA than Southern blot RFLP analysis and is less time-consuming. For example. In the third step. This enzyme. typically repeated 5-100 times.1998. known as annealing. PCR is performed in a thermal cycler.constitute one PCR “cycle”. the sample is cooled to 40°-65°C. On the other hand. PCR amplification (Figure 5) uses an enzyme known as Taq polymerase. In the first step of the PCR reaction. A VNTR is a region that is variably composed of a 15-70 base pair sequence. the chances of two people chosen at random having the same pattern is enormously remote. Also included in the PCR reaction mixture are two synthetic oligonucleotides known as “primers” and the extracted DNA. This document. This will result in one or two DNA bands corresponding to one or both chromosome segments recognized. Inc. It should be kept in mind that the total human population on earth is between 5-6 billion. 2004. and extension . The single-locus probes (SLPs) which detect a single segment of the repetitive DNA located at a specific site on a single chromosome.

or any part. in conjunction with use of accompanying reagents. is permitted for classroom/laboratory use only.1998.edvotek. 2006. Inc. EDVOTEK. 2004.com .. This document. Inc. Copyright © 1994.1995. 2000.1997. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.DNA Paternity Testing Simulation EDVO-Kit #  114 DNA Paternity Testing Target Sequence 5' 3' 3' 5' = Separation of two DNA strands = Primer 1 = Primer 2 Background Information 5' 3' 3' Denature 94°C 5' Cycle 1 5' 3' 5' 5' 3' 5' Anneal 2 primers 45°C 3' 5' 5' 5' 5' 3' Extension 72°C Cycle 2 5' 3' 3' 5' 5' 5' 5' 5' 5' 3' 5' 3' 5' 3' 5' 5' 3' 5' 5' 5' 5' 5' 3' 5' 5' 3' 5' 3' 5' 3' 5' 3' Cycle 3 5' 3' 5' 3' 3' 5' Figure 5: Amplification of DNA by Polymerase Chain Reaction Duplication of this document. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.

2006. Inc. This document. Background Information In this simulation experiment. DNA was extracted from samples obtained from the mother. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. investigators obtain a unique DNA profile for that individual which is unlike that of any other person (except for identical twins). or any part. THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.1997. The objective is to analyze and match the DNA fragment patterns after agarose gel electrophoresis and determine if Father 1 or Father 2 is the biological parent of the child.1998.. 2000.10 EDVO-Kit # 114 DNA Paternity Testing DNA Paternity Testing Simulation only 2-4 nucleotides in length.com . Copyright © 1994. Duplication of this document. in conjunction with use of accompanying reagents. By examining several different VNTRs or STRs from the same individual.1995. is permitted for classroom/laboratory use only.edvotek. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. EDVOTEK. 2004. child and two possible fathers. Inc.

may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. LABOrATOry SAFETy 1.edvotek. 2004. 4. 2. Gloves and goggles should be worn routinely as good laboratory practice. Copyright © 1994. Before starting the Experiment: • • Write a hypothesis that reflects the experiment. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory. 2000. in conjunction with use of accompanying reagents.. 5. or any part. or photograph the results. 3. During the Experiment: • Record (draw) your observations.1998.DNA Paternity Testing Simulation EDVO-Kit # 11 114 Experiment Overview and general Instructions ExPErImENT OBjECTIVE: The objective of this experiment module is to introduce students to the use of DNA Fingerprinting in a hypothetical paternity determination. Following the Experiment: • • • Formulate an explanation from the results. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. Write a hypothesis that would reflect this change. Predict experimental outcomes. Exercise caution when using any electrical equipment in the laboratory.1995. This document. is permitted for classroom/laboratory use only. Inc. Determine what could be changed in the experiment if the experiment were repeated.USE PIPET PUMPS.com . Duplication of this document. 2006. The Experiment LABOrATOry NOTEBOOK rECOrDINgS: Address and record the following in your laboratory notebook or on a separate worksheet. Inc. DO NOT MOUTH PIPET REAGENTS .1997. EDVOTEK. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents.

Duplication of this document.edvotek.com e .1998.connect leads to power source and conduct electrophoresis After electrophoresis..1 EDVO-Kit # 114 DNA Paternity Testing Simulation Experiment Overview and general Instructions ExPErImENT OVErVIEw: FLOw ChArT 1 2 The Experiment Remove end blocks & comb. is permitted for classroom/laboratory use only. Copyright © 1994. then submerge gel under buffer in electrophoresis chamber Prepare agarose gel in casting tray 3 A B C D E Load each sample in consecutive wells F 4 Attach safety cover. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. This document. transfer gel for staining 5 6 Analysis on white light source InstaStain® Methylene Blue aS Inst Blu et ®M tain Gel pattern will vary depending upon experiment. EDVOTEK. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. 2004. Inc. 2000.1997.1995. or any part. 2006. in conjunction with use of accompanying reagents. Inc.

you may be required to withdraw the appropriate amount of sample from the experiment stock tubes.edvotek. Alternatively.1997. make sure the entire volume of sample is at the bottom of the tubes before starting to load the gel. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.5 ml or 0. Inc. or any part. Sometimes a small amount of sample will cling to the walls of the tubes.DNA Paternity Testing Simulation EDVO-Kit # 1 114 Experiment Overview and general Instructions ABOuT ThE 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 or Individual 1. Check the sample volume. This document.. A protective overlay covers the strip of QuickStrip™ sample tubes. Briefly centrifuge the sample tubes. 2000. is permitted for classroom/laboratory use only.5 ml microtest Tubes • Your instructor may have aliquoted samples into a set of tubes for each lab group.com .1995. 2004. • • QuickStrips patent pending Duplication of this document. or tap the entire QuickStrip™ on the table to make samples fall to the bottom of the tubes. EDVOTEK QuickStrips™ A B C D E F • • Tap the overlay cover on top of the strip. Sometimes a small amount of sample will cling to the walls of the tubes. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. Inc. 2006.1998.5 ml or 0. EDVOTEK. Individual 1. make sure the entire volume of sample is at the bottom of the tubes before starting to load the gel. Check the sample volume. in conjunction with use of accompanying reagents.5 ml microtest tubes The Experiment Pre-aliquoted QuickStrip™ Connected Tubes • Each set of QuickStrip™ connected tubes contains pre-aliquoted ready-to-load samples for one gel. or tap each tube on the tabletop to get all the sample to the bottom of the tube. Copyright © 1994.

2. Note: The agarose gel is sometimes called a "submarine gel" because it is submerged under buffer for sample loading and electrophoretic separation. place it under buffer in an electrophoresis apparatus chamber. is permitted for classroom/laboratory use only. EDVOTEK electrophoresis experiments contain practice gel loading solution for this purpose.1995. One suggested activity is outlined below: 1. Pipeting mistakes can cause the sample to become diluted with buffer. to help control the delivery of small sample volumes. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. Follow guidelines below for delivering DNA samples to be stained with InstaStain® Methylene Blue.. in conjunction with use of accompanying reagents. After the gel solidifies. Take care not to damage or puncture the wells with the pipet tip. it is recommended that you practice sample delivery techniques before conducting the actual experiment. 3. Copyright © 1994. or any part. Variable automatic micropipet: Load the sample well with 35-38 microliters of sample. It is not necessary to prepare fresh buffer. shallow tray and submerge it under buffer or water. instead of the bulb. 2006. Transfer pipet: Load each sample well until it is full. Alternatively.14 EDVO-Kit # 114 DNA Paternity Testing Simulation Experiment Overview and general Instructions PrACTICE SAmPLE DELIVEry (gEL LOADINg) Accurate sample delivery technique ensures the best possible gel results. This document. Replace the practice gel with a fresh gel for the actual experiment.1997. the small amount of practice gel loading solution delivered to the wells will not interfere with the experiment. EDVOTEK.edvotek. gently squeeze the pipet stem. Cast a gel with the maximum number of wells possible. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. 5. Note: If practice gel loading is performed in the electrophoresis chamber. The Experiment If you are unfamiliar with loading samples in agarose gels. It is highly recommended that a separate agarose gel be cast for practice sample delivery. Inc. 2000. If you are using a: If you are using transfer pipets.com . • • 4. Duplication of this document.1998. Practice delivering the practice gel loading solution to the sample wells. 2004. Inc. If you need more practice. your teacher may have cut the gel into sections between the rows of wells. remove the practice gel loading solution by squirting buffer into the wells with a transfer pipet. Place a gel section with wells into a small. or cause damage to the wells with the pipet tip while loading the gel.

4. Inc.1997. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. 2004. 7. in conjunction with use of accompanying reagents. or any part.DNA Paternity Testing Simulation EDVO-Kit # 15 114 Experiment Overview and general Instructions gEL LOADINg TIPS FOr QuICKSTrIP™ SAmPLES Pipetting with micropipets The Experiment 1. Delivering QuickStrip™ Samples with Transfer Pipets: If using disposable transfer pipets for sample delivery. gently tap the QuickStrip™ on the lab bench to concentrate the sample to the bottom of the tube. EDVOTEK. Note: If a sample becomes displaced while inserting the pipet tip in the tube.1998. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. This document. Copyright © 1994. Gently pierce the printed protective overlay with a fresh pipet tip attached to a micropipet. using a fresh pipet tip for each sample. 2000.edvotek. 5. Discard the tip. Depress the micropipet plunger to the first stop before the tip is placed in contact with the sample. Raise the plunger of the micropipet to withdraw the sample.com . insert the tip into the sample. pierce the protective overlay with a paper clip before inserting the transfer pipet to withdraw the sample. With the pipet plunger depressed to the first stop. Stabilize the QuickStrip™ by firmly anchoring it on the lab bench. Gently tap the QuickStrip™ tubes on the lab bench to ensure that samples are at the bottom of the tubes. 2006.1995. Load the sample into the appropriate well of the gel. Duplication of this document. Repeat steps 3-6. Do not disturb the samples in the QuickStrip™. Inc. 2. is permitted for classroom/laboratory use only. 3. With the pipet plunger depressed to the first stop. 6. re-insert the tip into the sample and raise the micropipet plunger to withdraw the sample..

Make sure the comb sits firmly and evenly across the bed. extend the tape over the sides and bottom edge of the bed. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.1 EDVO-Kit # 114 DNA Paternity Testing Simulation Electrophoresis ..1995. 2. 2006. Duplication of this document. A. or any part. Press contact points firmly to form a good seal. This document. 2000. EDVOTEK. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.1998.Agarose gel Preparation AgArOSE gEL rEQuIrEmENTS FOr ThIS ExPErImENT • • Recommended gel size: Number of sample wells required: Placement of well-former template: Agarose gel concentration: 7 x 7 cm or 7 x 14 cm 5 First set of notches 0. Taping with labeling or masking tape: • • With 3/4 inch wide tape.com . in conjunction with use of accompanying reagents. Inc. Place a well-former template (comb) in the first set of notches at the end of the bed. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape. B.8% The Experiment • • PrEPArINg ThE gEL BED 1. Using Rubber dams: • Place a rubber dam on each end of the bed. 2004. is permitted for classroom/laboratory use only.1997.edvotek. Inc. Copyright © 1994. Fold the extended edges of the tape back onto the sides and bottom. Make sure the rubber dam fits firmly in contact with the sides and bottom of the bed.

8% If preparing the gel with diluted (1x) buffer.1998.1995. EDVOTEK. in conjunction with use of accompanying reagents.46 0.8 30 60 *0. indicate the level of the solution volume on the outside of the flask. 2006.75% UltraSpec-Agarose™ gel percentage rounded up to 0.6 1.1 or A.DNA Paternity Testing Simulation EDVO-Kit # 1 114 Electrophoresis . is permitted for classroom/laboratory use only.Agarose gel Preparation CASTINg ThE AgArOSE gEL(S) 3. 2000. 2) below. Use a 250 ml flask or beaker to prepare the gel solution. See Table B. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. Swirl the mixture to disperse clumps of agarose powder. IMPORTANT: Check with your instructor regarding the concentration of the buffer you are using to prepare your gel.8%* UltraSpec-Agarose™ Gel DNA Staining with InstaStain® MetBlue Size of Gel (cm) Amt of Agarose (g) + Distilled Total Concentrated Buffer (50x) + Water = Volume (ml) (ml) (ml) 7x7 7 x 14 0.1 Table Individual 0.edvotek. use Table A. 2004. Use the appropriate table (A. This document. 5. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.2 29. Diluted buffer is 1 volume of concentrated buffer to every 49 volumes of distilled or deionized water. Duplication of this document.4 58.2. 4.1997. Copyright © 1994.com .23 0. The Experiment If preparing the gel with concentrated (50x) buffer. A. or any part. Inc.. use Table A.1. Inc. With a marking pen.

continue with step 8. If detectable evaporation has occurred. Inc.com . The Experiment Hot plate method: • • Cover the flask with aluminum foil to prevent excess evaporation. 2006. 10. Inc.1995. Duplication of this document. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. go to step .edvotek. Copyright © 1994.18 EDVO-Kit # 114 DNA Paternity Testing Simulation Electrophoresis . 7. 2000. Cover the flask with plastic wrap to minimize evaporation. Heat the mixture to boiling over a burner with occasional swirling. or any part. Swirl the mixture and heat on High in bursts of 25 seconds until all the agarose is completely dissolved.gel Preparation 6. • • Use a transfer pipet to deposit a small amount of cooled agarose to both inside ends of the bed. Cool the agarose solution to 60°C with careful swirling to promote even dissipation of heat. EDVOTEK. Pour the cooled agarose solution into the bed. is permitted for classroom/laboratory use only. DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED.1997. Allow the gel to completely solidify. 60˚C After the gel is cooled to 0°C: If you are using rubber dams. 2004. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. It will become firm and cool to the touch after approximately 20 minutes. If you see "crystal" particles.. 9. Check the solution carefully. in conjunction with use of accompanying reagents. add distilled water to bring the solution up to the original volume marked on the flask in step 6. The final solution should appear clear (like water) without any undissolved particles. Hot agarose solution may irreversibly warp the bed. Heat the mixture to dissolve the agarose powder.1998. the agarose is not completely dissolved. Wait approximately 1 minute for the agarose to solidify. Boil until all the agarose is completely dissolved. Seal the interface of the gel bed and tape to prevent the agarose solution from leaking. A. it is recommended to use a microwave oven to reach boiling temperatures. If you are using tape. 8. Microwave method: At high altitudes. Heat the mixture on High for 1 minute. This document. Make sure the bed is on a level surface. • • • B.

A thin plastic knife. Place the gel (on its bed) into the electrophoresis chamber.. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer. After the gel is completely solidified. 13. Pour the appropriate amount of 1x buffer into the electrophoresis chamber according to Table B below.com . 2004. pH 7. Copyright © 1994. Remove the comb by slowly pulling straight up.edvotek. properly oriented. IMPORTANT: Check with your instructor to determine if the buffer has previously been diluted. Inc. Be especially careful not to damage or tear the gel wells when removing the rubber dams.gel Preparation PrEPArINg ThE gEL FOr ELECTrOPhOrESIS 11. 12.8. Prepare buffer as required for your electrophoresis unit. centered and level on the platform. the recommended electrophoresis buffer is Tris-acetate-EDTA. Buffer (ml) 6 8 10 20 EDVOTEK Model # Dilution + Water (ml) 294 392 490 980 Distilled M6+ M12 M36 (blue) M36 (clear) 300 400 500 1000 15.1995. The formula for diluting EDVOTEK (50x) concentrated buffer is 1 volume of buffer concentrate to every 49 volumes of distilled or deionized water. in conjunction with use of accompanying reagents. 2000. Make sure that the gel is completely submerged under buffer before proceeding to loading the samples and conducting electrophoresis. The Experiment For DNA analysis. spatula or pipet tip can be inserted between the gel and the dams to break possible surface tension. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.DNA Paternity Testing Simulation EDVO-Kit # 1 114 Electrophoresis . 2006. 14.1997. carefully and slowly remove the rubber dams or tape from the gel bed. or any part. is permitted for classroom/laboratory use only. EDVOTEK. Inc.1998. Table B Electrophoresis (Chamber) Buffer Total Volume Required (ml) 50x Conc. Do this carefully and evenly to prevent tearing the sample wells. This document. Duplication of this document.

Black Sample wells – Red + ruNNINg ThE gEL 1. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. 2006. or any part. the DNA samples migrate through the agarose gel towards the positive electrode. samples should be loaded into the wells of the gel in consecutive order..edvotek. Insert the plug of the red wire into the red input of the power source (positive input). Add a small amount of distilled water to the affected sample(s) until all tubes in the strip appear to contain the same total volume. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. Lane 1 2 3 4 5 Tube A B C D E Standard DNA Fragments Mother DNA cut with Enzyme Child DNA cut with Enzyme Father 1 DNA cut with Enzyme Father 2 DNA cut with Enzyme The Experiment Reminder: During electrophoresis.com . The amount of sample that should be loaded is 35-38 µl. make sure the gel is properly oriented in the apparatus chamber. in conjunction with use of accompanying reagents. This document. Inc. Before loading the samples. 2004. Copyright © 1994. After the DNA samples are loaded. Load the DNA samples into the wells in consecutive order.0 EDVO-Kit # 114 DNA Paternity Testing Simulation Electrophoresis . Insert the plug of the black wire into the black input of the power source (negative input). carefully snap the cover down onto the electrode terminals. EDVOTEK. is permitted for classroom/laboratory use only. Make sure that the negative and positive color-coded indicators on the cover and apparatus chamber are properly oriented.1997.1998. 2. 2000.Conducting Electrophoresis On occasion.1995. Inc. LOAD ThE SAmPLES For either QuickStrip™ or individual microtest tube format. evaporation may cause a QuickStrip™ sample to become more concentrated (sample appears to have lost some volume). Duplication of this document.

turn off the power. Check with your instructor regarding which staining method you should use. Copyright © 1994. Table C Time and Voltage Recommendations EDVOTEK Electrophoresis Model The Experiment Volts M6+ Minimum / Maximum M12 & M36 Minimum / Maximum 150 125 70 50 15 / 20 min 20 / 30 min 35 / 45 min 50 / 80 min 25 / 35 min 35 / 45 min 60 / 90 min 95 / 130 min 4. Instructions for a third staining option using liquid Methylene Blue Plus™ is also provided. 2004.edvotek. or any part.1995. unplug the power source.1998. Two options are provided for using the InstaStain® Methylene Blue cards. Method 1: One-step Staining and Destaining with InstaStain® MetBlue Method 2: Staining with InstaStain® Methylene Blue Method 3: Liquid Staining with Methylene Blue Plus Duplication of this document. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. ABOuT DNA gEL STAININg After electrophoresis. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. EDVOTEK.1997. Inc.. 5. Check to see that current is flowing properly - you should see bubbles forming on the two platinum electrodes. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor.DNA Paternity Testing Simulation EDVO-Kit # 1 114 Electrophoresis . Remove the gel from the bed for staining. in conjunction with use of accompanying reagents.Conducting Electrophoresis 3. General guidelines are presented in Table C. the agarose gels require staining in order to visualize the separated DNA samples. This experiment features a proprietary stain called InstaStain® Methylene Blue. Inc.com . After the electrophoresis is completed. is permitted for classroom/laboratory use only. 2000. 6. disconnect the leads and remove the cover. This document. 2006.

or can be left overnight.1997. Inc. 1. in conjunction with use of accompanying reagents. clean tray containing 75 ml of distilled or deionized water. or small plastic food containers 2. One Step Stain and Destain aSta Inst in™ Examples of small trays include large weigh boats. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. the gel is ready for visualization and photography. After staining and destaining. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. 2004. 2000. 3. This one-step method can be completed in approximately 3 hours. The agarose gel should be completely covered with liquid. Inc. The Experiment Wear gloves and safety goggles Do not stain gel(s) in the electrophoresis apparatus. STOrAgE AND DISPOSAL OF INSTASTAIN® mEThyLENE BLuE CArDS AND gELS • Stained gels may be stored in the refrigerator for several weeks.edvotek. or used electrophoresis buffer.com . DO NOT FREEZE AGAROSE GELS! • • Used InstaStain® cards and destained gels can be discarded in solid waste disposal. Gently float a 7 x 7 cm card of InstaStain® MetBlue with the stain side (blue) facing the liquid.1995. Duplication of this document.. Destaining solutions can be disposed down the drain. Copyright © 1994. 4. The gel can be left in the liquid overnight (cover with plastic wrap to prevent evaporation). or any part. EDVO-Kit # 114 DNA Paternity Testing Simulation Electrophoresis . Let the gel soak undisturbed in the liquid for approximately 3 hours. EDVOTEK.1998. Place the gel in a sealable plastic bag with destaining liquid.Staining and Visualization of DNA mEThOD 1: ONE-STEP STAININg AND DESTAININg wITh INSTASTAIN® mEThyLENE BLuE Agarose gels can be stained and destained in one easy step with InstaStain™ Methylene Blue cards. Remove the 7 x 7 cm agarose gel from its bed and completely submerse the gel in a small. is permitted for classroom/laboratory use only. This document. 2006.

Staining and Visualization of DNA mEThOD : STAININg wITh INSTASTAIN® mEThyLENE BLuE CArDS 1. • Add approximately 100 ml of distilled water to cover the gel. place the blue dye side of the InstaStain® Methylene Blue card on the gel. 6.DNA Paternity Testing Simulation EDVO-Kit #  114 Electrophoresis . Destain with 37°C distilled water. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. Allow the InstaStain® Methylene Blue to sit on the gel for 5 to 10 minutes. To ensure continuous contact between the gel and the InstaStain® card. After staining. place a gel casting tray and weight. in conjunction with use of accompanying reagents. This document. Patents Pending. 3 DNA InstaSt ain™ Patents Pendin g 5. Inc. 2000. on top of the InstaStain® card. Copyright © 1994.. Transfer the gel to a large weigh boat or small plastic container. Duplication of this document. After electrophoresis. remove the InstaStain® card. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. Press firmly. 5 minutes.1995. 2006. Destaining and Visualization of DNA 7. 8. Destain with distilled water (see “Destaining Notes” on following page). 2004. 6 InstaStain is a registered trademark of EDVOTEK. Inc. or any part.edvotek. 2. 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. g Patents Pendin Place a small weight for approx. Inc.com . such as a small empty beaker. 4. in g Pat Place the InstaStain® card on the gel. Wearing gloves. 1 Place gel on a flat surface covered with plastic wrap. is permitted for classroom/laboratory use only.1997. EDVOTEK. 5 Transfer to a small tray for destaining. The Experiment Wear gloves and safety goggles 2 aSta Inst in™ nd s Pe ent 3. 4 InstaStain™ - If the color of the gel appears very light. place the agarose gel on a flat surface covered with plastic wrap. wet the gel surface with buffer or distilled water and place the InstaStain® card back on the gel for an additional 5 minutes.1998.

DO NOT EXCEED 37°C ! Warmer temperatures will soften the gel and may cause it to break. Excessive destaining will cause the bands to be very light. Duplication of this document. 2006. or any part. 2000.1997.1998. EDVOTEK. This document. the larger DNA bands will become sharper and the smaller bands will be visible.edvotek. Copyright © 1994. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. repeat the staining and destaining procedures.1995. 11. DESTAININg NOTES: • • • Warmed distilled water at 37°C will accelerate destaining. Inc. DO NOT FREEZE AGAROSE GELS! • • Used InstaStain® cards and destained gels can be discarded in solid waste disposal. To optimize visibility.com . The volume of distilled water for destaining depends upon the size of the tray. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. • STOrAgE AND DISPOSAL OF INSTASTAIN® mEThyLENE BLuE CArDS AND gELS • Stained gels may be stored in the refrigerator for several weeks. Inc.Staining and Visualization of DNA 9. the entire background will become uniformly light blue. in conjunction with use of accompanying reagents.4 EDVO-Kit # 114 DNA Paternity Testing Simulation Electrophoresis .. Use the smallest tray available that will accommodate the gel. is permitted for classroom/laboratory use only. With additional destaining. The Experiment The larger DNA bands will initially be visible as dark blue bands against a lighter blue background. Destaining solutions can be disposed down the drain. The gel should be completely submerged during destaining. 10. When the gel is completely destained. Carefully remove the gel from the destain solution and examine the gel on a Visible Light Gel Visualization System. 2004. If the gel is too light and bands are difficult to see. use the amber filter provided with EDVOTEK equipment. Destaining will take longer with room temperature water. Do not exceed 3 changes of water for destaining. Place the gel in a sealable plastic bag with destaining liquid. Repeat destaining by changing the distilled water as needed.

2000. STOrAgE AND DISPOSAL OF STAIN AND gEL • Gels stained with Methylene Blue Plus™ may be stored in the refrigerator for several weeks. You may also leave the gel(s) in destain overnight.Staining and Visualization of DNA mEThOD : LIQuID STAININg wITh mEThyLENE BLuE PLuS™ Dilution of methylene Blue Plus™ Stain • Dilute the 10x stain by mixing 1 part stain with 9 parts distilled or deionized water. Inc. Inc. 3. Destain in 600 ml of distilled water that has been warmed to 37°C.DNA Paternity Testing Simulation EDVO-Kit # 5 114 Electrophoresis . DO NOT EXCEED 37°C ! Warmer temperatures will soften the gel and may cause it to break.edvotek. The Experiment Wear gloves and safety goggles Do not stain gel(s) in the electrophoresis apparatus. Place the gel in a sealable plastic bag with destaining liquid. is permitted for classroom/laboratory use only. 4.1997. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.1995. 2006. EDVOTEK. with occasional agitation. 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. • Bands will become visible after the second destain. or any part. If the gel is too light and bands are difficult to see.. Carefully remove the gel from the destain solution and examine on a Visible Light Gel Visualization System. Staining and Destaining 1. 5. Each group should mark their gel. Duplication of this document.1998. in conjunction with use of accompanying reagents. To optimize visibility. • Stained gels which are not kept can be discarded in solid waste disposal. This document. Copyright © 1994. • Completely submerse the gel(s) in 600 ml of 37°C distilled water for 15 minutes with occasional agitation. 2004. Methylene Blue Plus™ stain and destaining solutions can be disposed down the drain. Stain gel(s) for a minimum of 30 minutes. or making a small hole in a designated corner.com . Then discard the destaining solution Change the distilled water for a second destain for another 15 minutes with occasional agitation. use the amber filter provided with EDVOTEK equipment. 2. DO NOT FREEZE AGAROSE GELS. Do not stain gel(s) in the electrophoresis apparatus. to facilitate identification after staining and destaining. such as removing a small slice. repeat the staining and destaining procedures. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.

This document.1998. Why do different individuals such as siblings have different restriction enzyme recognition sites? What is the function of PCR primers used in DNA paternity analysis? Why is there more than one single locus used in an actual paternity DNA test? Why do we not use probes in this DNA paternity simulation and still obtain results? The Experiment 3. EDVOTEK. Inc. Inc. or any part. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. 2000. 2006.1995. EDVO-Kit # 114 Study Questions DNA Paternity Testing Simulation Answer the following study questions in your laboratory notebook or on a separate worksheet. 2. Duplication of this document.. 4. Copyright © 1994. 1. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. 2004.com .1997. is permitted for classroom/laboratory use only. in conjunction with use of accompanying reagents.edvotek.

Analysis of the experiments will provide students the means to transform an abstract concept into a concrete explanation. EDuCATIONAL rESOurCES Visit our web site for information about EDVOTEK's complete line of experiments for biotechnology and biology education. even the most experienced students and teachers occasionally encounter experimental problems or difficulties. Eastern time zone. For example. TECH VO Mon . EVT 007206AM .com email: edvotek@aol. In addition. These guidelines can be adapted to fit your specific set of circumstances. NATIONAL CONTENT AND SKILL STANDArDS Online Ordering now available By performing this experiment. a variety of resources are continuously being added to the EDVOTEK web site. please refer to the EDVOTEK website. Please visit our website for specific content and skill standards for various experiments. students will learn to load samples and run agarose gel electrophoresis.Fri 9 am Please have the following information ready: • Experiment number and title • Kit lot number on box or tube • Literature version number (in lower right corner) • Approximate purchase date EDVOTEK . The EDVOTEK web site provides several suggestions and reminders for conducting electroTechnical Service phoresis. 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.com FAx: (01) 40-058 • email: edvotek@aol. length of laboratory sessions. Electrophoresis hints.Fri ED Laboratory Extensions and Supplemental 9:00 am to 6:00 pm ET 1-800-EDVOTEK (1-800-338-6835) Activities Mo pm -6 n . For DNA Sizing instructions.edvotek.com ET FAX: (301) 340-0582 Web: www.com Laboratory extensions are easy to perform using EDVOTEK experiment kits. Technical Service is available from 9:00 am to 6:00 pm.edvotek. as well as answers to frequently asked Department S E RV I C E electrophoresis questions. If you do not find the answers to your questions in this section. and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. which is updated on a continuous basis with educational activities and resources. However. a DNA sizing determination activity can be performed on any electrophoresis gel result containing DNA markers run in parallel with other DNA samples.The Biotechnology Education Company® 1-800-EDVOTEK • www.DNA Paternity Testing Simulation EDVO-Kit #  114 Instructor’s guide Notes to the Instructor: Class size. Call for help from our knowledgeable technical staff at 1-800-EDVOTEK (1-800-338-6835). and other laboratory extension suggestions.

Table C Time and Voltage Recommendations EDVOTEK Electrophoresis Model Volts M6+ Minimum / Maximum M12 & M36 Minimum / Maximum 150 125 70 50 15 / 20 min 20 / 30 min 35 / 45 min 50 / 80 min 25 / 35 min 35 / 45 min 60 / 90 min 95 / 130 min 3. a larger quantity of UltraSpecAgarose can be prepared for sharing by the class. Generally. gel preparation: Whether you choose to prepare the gel(s) in advance or have the students prepare their own. 20 minutes of this time is required for gel solidification. Generally. Individual gel Casting: Each student lab group can be responsible for casting their own individual gel prior to conducting the experiment. Duplication of this document. learn and practice pipeting techniques by preparing and delivering various dye mixtures to a special Pipet Card™. the higher the voltage applied the faster the samples migrate. Casting of a separate practice gel is highly recommended. PrEPArINg AgArOSE gELS FOr ELECTrOPhOrESIS There are several options for preparing agarose gels for the electrophoresis experiments: 1. 2006. 2000. the maximum amount of voltage significantly depends upon the design of the electrophoresis apparatus and should not exceed manufacturer's recommendations. focuses exclusively on the use of micropipets.8 EDVO-Kit # 114 Notes to the Instructor: DNA Paternity Testing Simulation APPrOxImATE TImE rEQuIrEmENTS 1. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. 2.com . This activity can require anywhere from 10 minutes to an entire laboratory session.. Inc. Micropipetting Basics. To save time. allow approximately 30-40 minutes for this procedure. However. See instructions for "Batch Gel Preparation".edvotek. EDVOTEK electrophoresis experiments contain a tube of practice gel loading solution for this purpose.1998. is permitted for classroom/laboratory use only. Students 2. in conjunction with use of accompanying reagents.1995. Different models of electrophoresis units will separate DNA at different rates depending upon its configuration and the distance between the two electrodes. 2004.1997. Copyright © 1994. Batch gel Preparation: A batch of agarose gel can be prepared for sharing by the class. Inc. EDVOTEK. This document. Conducting Electrophoresis: The approximate time for electrophoresis will vary from approximately 15 minutes to 2 hours depending upon various factors. or any part. depending upon the skill level of your students. a practice activity is suggested prior to conducting the experiment. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. Instructor’s guide EDVOTEK Experiment # S-44. Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C. Practice gel Loading: If your students are unfamiliar with using micropipets and sample loading techniques.

should be "anchored" back to the tray with a few drops of hot. Solidified gels can be stored under buffer in the refrigerator for up to 2 weeks.1997. EDVOTEK. EDVOTEK also offers InstaStain® Ethidium Bromide (InstaStain® EtBr) and Protein InstaStain® for staining Protein polyacrylamide gels. or can be left in liquid overnight. • Gels that have been removed from their trays for storage. Inc.1995. 2000. Inc. Method 2. allow the gel to destain for several hours to overnight. Do not store gels at -20°C. This document. Freezing will destroy the gels. Preparing gels in Advance: • Gels may be prepared ahead and stored for later use. agarose gels can be stained and destained in one easy step. and will become sharper with additional destaining. which can be completed in approximately 3 hours. This will allow the stained gel to "equilibrate" in the destaining solution. or any part. Instructions for a third staining option using liquid Methylene Blue Plus™ is also provided. molten agarose before placing the gels into the apparatus for electrophoresis. 2006. DNA bands will become visible after destaining for approximately 20 minutes.DNA Paternity Testing Simulation EDVO-Kit #  114 Notes to the Instructor: 3. Copyright © 1994. Duplication of this document. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.edvotek. Two options are provided for using the InstaStain® Methylene Blue cards. This will prevent the gels from sliding around in the trays and the chambers.1998. It is a proprietary new staining method which saves time and reduces liquid waste. For the best photographic results. resulting in dark blue DNA bands contrasting against a uniformly light blue background. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. 2004. is permitted for classroom/laboratory use only. requires approximately 5-10 minutes for staining. in conjunction with use of accompanying reagents. Instructor’s guide gEL STAININg AND DESTAININg AFTEr ELECTrOPhOrESIS This experiment features InstaStain® Methylene Blue for gel staining after electrophoresis.com . Method 1: One-step Staining and Destaining with InstaStain® MetBlue Method 2: Staining with InstaStain® Methylene Blue Method 3: Liquid Staining with Methylene Blue Plus™ Using Method 1.. using InstaStain® Methylene Blue cards.

Before loading the samples.5 ml) microtest tubes FOrmAT: PrE-ALIQuOTED QuICKSTrIP™ CONNECTED TuBES A EDVOTEK® • DO NOT BEND A B C CUT HERE CUT HERE A B C CUT HERE A B C CUT HERE A B C CUT HERE A B C D E F G H B C D E F G H D E F G H D E F G H D E F G H D E F G H Convenient QuickStrip™ connected tubes contain pre-aliquoted ready-to-load samples.0 EDVO-Kit # 114 Pre-Lab Preparations DNA Paternity Testing Simulation rEADy-TO-LOAD DNA SAmPLES FOr ELECTrOPhOrESIS No heating required before gel loading. 2006. The samples are packaged in a microtiter block of tubes covered with a protective overlay. This document. Remind students to tap the tubes before gel loading to ensure that all of the sample is at the bottom of the tube. 1.com . Use sharp scissors to separate the block of samples into individual strips as shown in the diagram at left. Electrophoresis samples and reagents in EDVOTEK experiments are packaged in various formats.5 ml (or 0. Each group will require one strip of samples. 3. The samples in Series 100 and S-series electrophoresis experiments will be packaged in one of the following ways: 1) 2) On occasion.1998. Duplication of this document. 4. Carefully cut between each set of tubes Each row of samples (strip) constitutes a complete set of samples for each gel. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. is permitted for classroom/laboratory use only. Inc. Pre-aliquoted Quickstrip™ connected tubes OR Individual 1. EDVOTEK.edvotek. evaporation may cause a QuickStrip™ sample to become more concentrated (sample appears to have lost some volume).1995. Some tubes may be empty. The number of samples per set will vary depending on the experiment. A B C D E F 2. in conjunction with use of accompanying reagents. Do not cut or puncture the protective overlay directly covering the sample tubes. Instructor’s guide 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. 2000.1997.. Copyright © 1994. Series 100 experiments feature DNA samples which are predigested with restriction enzymes and are stable at room temperature. or any part. Cut carefully between the rows of samples. 2004. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. DNA samples are ready for immediate delivery onto agarose gels for electrophoretic separation and do not require pre-heating in a waterbath. Separate the microtiter block of tubes into strips for a complete set of samples for one gel. instruct students to add a small amount of distilled water to the affected sample(s) until all tubes in the strip appear to contain the same total volume. Inc.

Copyright © 1994. Custom bulk quantities are also available by request.5 ml individual microtest tubes be aliquoted for each gel. s rie Se Se rie s Aliquot the samples into appropriately labeled 0.5 mL mICrOTEST TuBES It is recommended that samples packaged in 1. 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). Instruct students to set their automatic micropipets to a volume that is 2 microliters less than the volume you have aliquoted. Before aliquoting.3 cm 3.7 cm Mix well by inverting and tapping the tubes several times. check all sample volumes for possible evaporation. Inc. They should centrifuge the samples tubes.1998. or tap the tubes on the tabletop.3 cm level for the C-Series Instructor’s guide 4.1995.com .1997. For gels to be stained with InstaStain® Methylene Blue: 38-40 µl of each sample B 2. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. 2006. Then add distilled water to slightly above the following level: 2. If needed.3 cm level for the B-Series 3. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. is permitted for classroom/laboratory use only. or any part. in conjunction with use of accompanying reagents. EDVOTEK.DNA Paternity Testing Simulation EDVO-Kit # 1 114 Pre-Lab Preparations FOrmAT: INDIVIDuAL 1..3 cm After checking sample volumes and determining that the samples are at their proper total volumes: 1. 2. 2000. tap or centrifuge the sample tubes.edvotek. Some suggestions are: • Remind students to make sure all of the sample is at the bottom of the tube before gel loading. 2004. This document.5 ml or 1. The samples will become more concentrated if evaporation has occurred. C • Duplication of this document.5 ml microtest tubes. Inc. Students might have difficulty retrieving the entire aliquoted volume of sample because some of it may cling to the side walls of the tubes.

indicate the level of solution volume on the outside of the flask. Heat the agarose solution as outlined previously for individual gel preparation. This document. Allow the gel to completely solidify. If the amount of agarose is not specified or if the bottle's plastic seal has been broken. It will become firm and cool to the touch after approximately 20 minutes. Duplication of this document.8% Note: The UltraSpec-Agarose™ kit component is often labeled with the amount it contains. Cool the agarose solution to 60°C with swirling to promote even dissipation of heat. in conjunction with use of accompanying reagents. With a marking pen. Please read the label carefully. 2006. 7.1995. *0. Use a 500 ml flask to prepare the diluted gel buffer 2.5 390 3. Inc. Instructor’s guide Table D Bulk Preparation of Electrophoresis Buffer Distilled Water (ml) = Total Volume (ml) BuLK ELECTrOPhOrESIS BuFFEr Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is outlined in Table D. The heating time will require adjustment due to the larger total volume of gel buffer solution. Inc. Dispense the required volume of cooled agarose solution for casting each gel. or any part. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.0 7. Amt of Agarose (g) 3. Then proceed with preparing the gel for electrophoresis.com . weigh the agarose to ensure you are using the correct amount.8% agarose gels. Swirl to disperse clumps. 4. see Table E. In many cases. 2000. Copyright © 1994.8%* UltraSpec-Agarose™ + Distilled Total Concentrated Buffer (50x) + Water = Volume (ml) (ml) (ml) 1. add distilled water to bring the solution up to the original volume as marked on the flask in step 3.0 grams.5 382. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. EDVO-Kit # 114 DNA Paternity Testing Simulation Quantity Preparations for Agarose gel Electrophoresis To save time. 2004.1998.edvotek.. 60˚C 6.8%) For quantity (batch) preparation of 0. the entire contents of the bottle is 3. Concentrated Buffer (50x) + (ml) 60 Table 2.940 3000 (3 L) BATCh AgArOSE gELS (0. Unused diluted buffer can be used at a later time and solidified agarose gel solution can be remelted. If evaporation has occurred. the electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class. 5. E Batch Preparation of 0.0 grams of UltraSpec-Agarose™ into the prepared buffer. is permitted for classroom/laboratory use only. Pour 3. EDVOTEK. The volume required is dependent upon the size of the gel bed.75% UltraSpec-Agarose™ gel percentage rounded up to 0.1997.

the relative positions of DNA fragments are shown but are not depicted to scale. 100 pages Duplication of this document.1998. # 1401 Cat. Inc. 2000. or visit the EDVOTEK web site at www. Copyright © 1994. or any part. EDVOTEK offers two student laboratory notebooks. in conjunction with use of accompanying reagents. Standard DNA Fragments Mother DNA cut with Enzyme Child DNA cut with Enzyme Father 1 DNA cut with Enzyme Father 2 DNA cut with Enzyme Laboratory notebooks It is highly recommended that students maintain a laboratory notebook to formulate hypotheses and to record experimental procedures and results. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. This document.1995. 2006. Cat..com.edvotek. Lane 1 2 3 4 5 Tube A B C D E In the idealized schematic. Inc. # 1402 Laboratory Notebook. is permitted for classroom/laboratory use only.edvotek. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. refer to the EDVOTEK Catalog listing for this experiment. 2004.com .1997.DNA Paternity Testing Simulation EDVO-Kit #  114 Experiment results and Analysis Instructor’s guide To view a color photo of the gel result. EDVOTEK. 50 pages Laboratory Notebook. Call 1-800-EDVOTEK (1-800-338-6835) for classroom quantity pricing information.

why is there more than one single locus used in an actual paternity DNA test? More than one locus is used to assure the test is determining several allelic differences in an individual that can be correlated to the composite summation of both the mother and father. .edvotek. Instructor’s guide . Chromosomes which are matched pairs are derived as one from the father and second from the mother. or any part. in conjunction with use of accompanying reagents. all rights reserved EVT 007206AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. 2000. EDVOTEK. Duplication of this document.1998..1995. The two copies of a gene at a specific locus of the chromosome represents the unique genotype of the offspring.1997. is permitted for classroom/laboratory use only. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.com . These differences can by directly compared by visualization without the use of probes used in Southern blot analysis. what is the function of PCr primers used in DNA paternity analysis? Two PCR primers are used for the amplification of a specific gene or DNA target. This document. Alleles are a result of genetic traits which can be dominant or recessive. 4. Inc. Copyright © 1994. Each primer binds specifically to one of the DNA strands at 5’ or 3’ ends of the gene and synthesize the opposite DNA strand using the Taq DNA polymerase and the 4 deoxynucleotide triphosphates.4 EDVO-Kit # 114 DNA Paternity Testing Simulation Study Questions and Answers 1. why do we not use probes in this DNA paternity simulation and still obtain results? In this experiment. 2006. we use genetically engineered plasmids that yield a very limited number of bands corresponding to simulation of allelic genes. The two genes can be alleles which result in yielding a unique restriction enzyme digest pattern for that gene. why do different individuals such as siblings have different restriction enzyme recognition sites? There are a large number of alleles of a gene. Inc. 2004.

or no information is available.D. skin. Material Safety Data Sheet Section V . N. no odor N. eyes None Skin: Wash with soap and water Other Limits Recommended Medical Conditions Generally Aggravated by Exposure % (Optional) Emergency First Aid Procedures This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard.D.Physical/Chemical Characteristics Flash Point (Method Used) Extinguishing Media Special Fire Fighting Procedures N. liquid.Physical/Chemical Characteristics Flash Point (Method Used) Extinguishing Media No data Flammable Limits N. Special Water spray.EDVOTEK Agarose ® May be used to comply with OSHA's Hazard Communication Standard. UEL Section VIII . Ingestion: If conscious. carbon dioxide.Reactivity Data Stability Incompatibility Unstable Stable Conditions to Avoid X None No data available IDENTITY (As Used on Label and List) Hazardous Decomposition or Byproducts Section I Manufacturer's Name Emergency Telephone Number Telephone Number for information Date Prepared Hazardous Polymerization Route(s) of Entry: May Occur Will Not Occur Conditions to Avoid EDVOTEK. Zip Code) (301) 251-5990 (301) 251-5990 07/01/03 Section VI . If any item is not applicable. Common Name(s)] OSHA PEL ACGIH TLV Irritation to upper respiratory tract. slight vinegar odor Other Precautions Avoid eye and skin contact.Health Hazard Data Health Hazards (Acute and Chronic) Carcinogenicity: X Yes None Skin? Inhalation? Yes Ingestion? Yes 14676 Rothgeb Drive Rockville. or no information is available. Street. Dispose in accordance with all applicable federal. or collect in absorptive material and dispose of the absorptive material.Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method No data No data No data Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1) No data No data No data Wear suitable protective clothing. Street.1200 Standard must be consulted for specific requirements. Mop up spill and rinse with water. Wear protective equipment and SCBA with full facepiece operated in positive pressure mode. dry chemical. and local enviromental regulations.Health Hazard Data Health Hazards (Acute and Chronic) Carcinogenicity: None identified Signs and Symptoms of Exposure X Yes None Skin? Yes Ingestion? Yes 14676 Rothgeb Drive Rockville. MD 20850 07/01/03 Inhalation: No data available NTP? Ingestion: Large amounts may cause diarrhea IARC Monographs? Signature of Preparer (optional) OSHA Regulation? Signs and Symptoms of Exposure Section II .) Vapor Density (AIR = 1) Solubility in Water Appearance and Odor Section VII . give large amounts of water Eyes: Flush with water Inhalation: Move to fresh air Section III .Control Measures Respiratory Protection (Specify Type) Ventilation Protective Gloves Chemical cartridge respirator with full facepiece. If any item is not applicable.cold White powder. Section VIII . Material Safety Data Sheet Section V . the space must be marked to indicate that. Inc. (greater than 10%) Clear.Physical/Chemical Characteristics Boiling Point Vapor Pressure (mm Hg. Precautions to be Taken in Handling and Storing Appreciable.Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity. = No data LEL No data Flammable Limits N.Control Measures N. Address (Number. CAS #9012-36-6 Treat symptomatically and supportively Section III . dilution ventilationOther Yes Eye Protection Splash proof goggles Unusual Fire and Explosion Hazards Other Protective Clothing or Equipment Work/Hygienic Practices Impervious clothing to prevent skin contact None EDVOTEK ® May be used to comply with OSHA's Hazard Communication Standard.Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method For 1% solution 194 F No data No data Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1) No data No data No data Sweep up and place in suitable container for disposal Normal solid waste disposal Precautions to be Taken in Handling and Storing Vapor Pressure (mm Hg.Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity. = No data LEL UEL None None Other Precautions Section IV . state. Carbon dioxide Conditions to Avoid IDENTITY (As Used on Label and List) Hazardous Decomposition or Byproducts 50x Electrophoresis Buffer Section I Manufacturer's Name Emergency Telephone Number Telephone Number for information Date Prepared Signature of Preparer (optional) Hazardous Polymerization Route(s) of Entry: May Occur Will Not Occur Inhalation? EDVOTEK. 29 CFR 1910.D. State.Physical/Chemical Characteristics Boiling Point Section VII . 29 CFR 1910. None identified Yes Yes Special Mechanical (General) Other None None Yes None None Eye Protection Safety goggles Unusual Fire and Explosion Hazards Other Protective Clothing or Equipment Work/Hygienic Practices . Common Name(s)] OSHA PEL ACGIH TLV No data available No data available Other Limits Recommended % (Optional) Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard.Reactivity Data Stability Incompatibility Unstable Stable Conditions to Avoid X None Strong oxidizing agents Carbon monoxide. City. Zip Code) (301) 251-5990 (301) 251-5990 Section VI . the space must be marked to indicate that.) Vapor Density (AIR = 1) Solubility in Water Appearance and Odor Insoluble .D. MD 20850 None NTP? IARC Monographs? OSHA Regulation? Section II . Inc.D. Respiratory Protection (Specify Type) Ventilation Protective Gloves Local Exhaust Use extinguishing media appropriate for surrounding fire.1200 Standard must be consulted for specific requirements. Address (Number. Note: Blank spaces are not permitted. Note: Blank spaces are not permitted. halon or standard foam Possible fire hazard when exposed to heat or flame None Local Exhaust Special Fire Fighting Procedures Mechanical (General)Gen. None Section IV .D. State. City.

7 Bis (Dimethylamino) Phenothiazin 5 IUM Chloride CAS # 61-73-4 No data available Treat symptomatically Section III .) Vapor Density (AIR = 1) Solubility in Water Appearance and Odor Section VII .82 No data available No data available IARC Monographs? Inhalation: Cyanosis OSHA Regulation? Section II .Health Hazard Data Health Hazards (Acute and Chronic) Carcinogenicity: X Yes None Skin? Yes Ingestion? Yes 14676 Rothgeb Drive Rockville. Common Name(s)] OSHA PEL ACGIH TLV May cause skin or eye irritation None reported Other Limits Recommended % (Optional) Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. Wear SCBA.EDVOTEK ® May be used to comply with OSHA's Hazard Communication Standard. Zip Code) (301) 251-5990 (301) 251-5990 Section VI . Treat symptomatically and supportively. Note: Blank spaces are not permitted. safety goggles Unusual Fire and Explosion Hazards Other Protective Clothing or Equipment Work/Hygienic Practices EDVOTEK ® May be used to comply with OSHA's Hazard Communication Standard. water spray or foam Yes Yes Special None None Splash proof goggles Special Fire Fighting Procedures Use agents suitable for type of surrounding fire.30420. Keep upwind. Address (Number. State. Material Safety Data Sheet Section V .Physical/Chemical Characteristics Flash Point (Method Used) Extinguishing Media No data Flammable Limits LEL No data UEL Section VIII . State.Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method No data No data No data Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1) No data No data No data Wear eye and skin protection and mop spill area. the space must be marked to indicate that. IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Section II . City.) Vapor Density (AIR = 1) Solubility in Water Appearance and Odor Section VII . Note: Blank spaces are not permitted. 29 CFR 1910.Physical/Chemical Characteristics Boiling Point Vapor Pressure (mm Hg. Street.Reactivity Data Stability Incompatibility Unstable Stable Conditions to Avoid X None Strong oxidizing agents IDENTITY (As Used on Label and List) Hazardous Decomposition or Byproducts InstaStain® Methylene Blue Toxic fumes of Carbon monoxide. sulfur oxides.Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method No data No data No data Soluble . no odor Avoid eye and skin contact. Precautions to be Taken in Handling and Storing Soluble Blue liquid. Address (Number. If any item is not applicable. carbon dioxide.cold Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1) No data No data No data Ventilate area and wash spill site Mix material with a combustible solvent and burn in chemical incinerator equipped with afterburner and scrubber. avoid breathing hazardous sulfur oxides and bromides.1200 Standard must be consulted for specific requirements.Physical/Chemical Characteristics Flash Point (Method Used) Extinguishing Media No data available Flammable Limits Section VIII . Section III . Rinse contacted area with copious amounts of water. Precautions to be Taken in Handling and Storing Keep tightly closed. Common Name(s)] OSHA PEL ACGIH TLV Other Limits Recommended % (Optional) Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Methylene Blue 3. dry place Other Precautions Chemical bound to paper. Inc. state. chloride gas May Occur Will Not Occur Inhalation? Conditions to Avoid Section I Manufacturer's Name Emergency Telephone Number Telephone Number for information Date Prepared Hazardous Polymerization Route(s) of Entry: EDVOTEK. no odor None LEL UEL Section IV . or no information is available.Reactivity Data Stability Incompatibility Unstable Stable Conditions to Avoid X None None Sulfur oxides. Inc. 29 CFR 1910. the space must be marked to indicate that. Street.Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity. City. dry chemical powder. Observe all federal.1200 Standard must be consulted for specific requirements.Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity. Unknown Other Yes None required Eye Protection Unusual Fire and Explosion Hazards Other Protective Clothing or Equipment Work/Hygienic Practices Avoid eye and skin contact . SCBA Special Water spray. carbon dioxide. or no information is available. Store in cool. and local regulations. Rinse with water. MD 20850 07/01/03 Skin: May cause skin irritation Eyes: May cause eye irritation NTP? Signature of Preparer (optional) Signs and Symptoms of Exposure Meets criteria for proposed OSHA medical records rule PEREAC 47. Material Safety Data Sheet Section V . Check local and state regulations. nitrogen oxides. MD 20850 07/01/03 Signature of Preparer (optional) No data available NTP? Acute eye contact: May cause irritation. No data available for other routes.Health Hazard Data Health Hazards (Acute and Chronic) Carcinogenicity: Yes Ingestion? Yes 14676 Rothgeb Drive Rockville. alcohol or polymer foam Local Exhaust Mechanical (General) Special Fire Fighting Procedures Required Other Self contained breathing apparatus and protective clothing to prevent contact with skin and eyes Emits toxid fumes under fire conditions Rubber Rubber boots Eye Protection Chem.Control Measures Respiratory Protection (Specify Type) Ventilation Protective Gloves Local Exhaust Mechanical (General) No data Dry chemical. None Other Precautions Section IV . Carbon dioxide.Physical/Chemical Characteristics Boiling Point Vapor Pressure (mm Hg. and bromides Conditions to Avoid IDENTITY (As Used on Label and List) Hazardous Decomposition or Byproducts Practice Gel Loading Solution Section I Manufacturer's Name Emergency Telephone Number Telephone Number for information Date Prepared Hazardous Polymerization Route(s) of Entry: May Occur Will Not Occur Inhalation? X Yes None Skin? EDVOTEK. Zip Code) (301) 251-5990 (301) 251-5990 Section VI . hydrogen. If any item is not applicable.Control Measures Respiratory Protection (Specify Type) Ventilation Protective Gloves No data No data MIOSH/OSHA approved.