DNA: A Person s Ultimate Fingerprint

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "DNA: A Person s Ultimate Fingerprint"

Transcription

1 A partnership between the UAB Center for Community Outreach Development and McWane Center DNA: A Person s Ultimate Fingerprint This project is supported by a Science Education Partnership Award (SEPA) from the National Center for Research Resources, National Institutes of Health. Revised 10-Sep-04

2 DNA: A Person s Ultimate Fingerprint The experiment described here is designed to allow students to examine individual variations in the human genome. Using polymerase chain reaction (PCR), a fingerprint of each member of the class will be made. DNA fragments generated by PCR will be separated by gel electrophoresis and analyzed for polymorphisms. Students will examine a single locus on chromosome 1, which is composed of non-coding repeats that vary in number from 14 to 41 in the human population. The experiment will allow students to isolate their own DNA from cheek cells, carry out PCR reactions and analyze the results of the PCR by gel electrophoresis. 2

3 Student Guide Human DNA Fingerprinting by Polymerase Chain Reaction In this experiment, polymerase chain reaction (PCR) is used to amplify a short nucleotide sequence from chromosome 1 to create a personal DNA fingerprint. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes that exhibit a great deal of diversity. Such variable sequences are termed polymorphic (meaning many forms) and provide the basis for genetic disease diagnosis and forensic/paternity testing. Many DNA polymorphisms are found within the estimated 90% of the human genome that does not code for protein. A special type of polymorphism called a VNTR (variable number of tandem repeats) is composed of a certain DNA sequence that is repeated and repeated and repeated, with each repeat lying adjacent to the next one. Chromosome 1 contains a VNTR called D1S80, which has a repeat unit of 16 base pairs. At the D1S80 locus, most individuals have alleles containing between 14 and 41 repeats, which are inherited in a Mendelian fashion on the maternal and paternal copies of chromosome 1. The source of template DNA is a sample of several thousand cheek cells obtained by saline mouthwash (bloodless and noninvasive). The cells are collected by centrifugation and resuspended with the resin Chelex, which binds metal ions that inhibit the PCR reaction. The cells are lysed by boiling and centrifuged to remove cell debris. A sample of the supernatant containing chromosomal DNA is combined with a buffered solution of heat-stable Taq polymerase, two oligonucleotide primers, the four deoxynucleotide building blocks of DNA, and the cofactor MgCl 2. The PCR mixture is placed in a DNA thermal cycler and taken through 30 cycles consisting of: 1 minute at 94 C chromosomal DNA is denatured into single strands 1 minute at 65 C primers anneal to their complementary sequences on either side of the D1S80 locus via hydrogen bonds 1 minute at 72 C Taq polymerase extends a complementary DNA strand from each primer The primers used in the experiment bracket the D1S80 locus and selectively amplify that region of chromosome 1. Following PCR amplification, student alleles are separated according to size using agarose gel electrophoresis. After staining with ethidium bromide, one or two bands are visible in each student lane, indicating whether an individual is homozygous or heterozygous for the D1S80 locus. Different alleles appear as distinct bands each composed of several billion copies of the amplified allele. A band s position in the gel indicates the size (and number of repeats) of the D1S80 allele: smaller alleles move a longer distance from their origin, while larger alleles move a shorter distance. 3

4 Laboratory Procedure Procedure A. Isolation of cheek cell DNA 1. Use a permanent marker to label your name on a test tube containing saline solution and on two clean 1.5 ml microfuge tubes. 2. Pour all of the saline solution (10 ml) into your mouth and vigorously swish for seconds. 3. Expel saline solution back into tube. 4. Using a P1000 micropipet, remove 1.5 ml of saline solution, now containing cheek cells, and add to one labeled microfuge tube. 5. Place a sample tube, together with other student samples, in balanced configuration in a microfuge and spin for 5 minutes at half maximum speed. 6. Carefully pour off supernatant into original test tube. Take care not to disturb cell pellet at the bottom of the microfuge tube. Remove any excess sup with a P100 or a tightly wrapped Kimwipe. Ask your facilitator for help! 7. Set micropipet to 500 µl. Draw Chelex suspension in and out of pipet tip several times to suspend resin beads. Then, before resin settles, rapidly transfer 500 µl of Chelex to the tube containing your cell pellet. 8. Resuspend cells by pipetting up and down several times. Examine against light to confirm that no visible clumps of cells remain. 9. Place your sample in a 95 C hot block for 10 minutes. Remove your tube from hot block and allow it to cool for a minute. 10. Place your sample tube in a balanced configuration in microcentrifuge, and spin for 30 seconds at maximum speed. 11. Use a fresh tip on the P100 micropipet to transfer 50 µl of the clear supernatant to the second labeled microfuge tube. Take care not to pick up Chelex/cell debris from the bottom of the tube. Discard tube with pellet of cells and Chelex. Begin Procedure B now (or store samples on ice until ready for Procedure B). 4

5 Procedure B. Set Up PCR Reaction: 1. Use permanent marker to label the cap of a PCR tube with your initials. Add the following reagents to the 0.2 ml PCR reaction tube: 37 µl Master Mix, (containing water, 10X Buffer and dntps) 3 µl D1S80 primers 5 µl cheek cell DNA 5 µl Taq polymerase (1u/µl) (Your facilitator will add the Taq. Make sure you watch it go in you PCR tube.) You should have a total reaction volume of 50µl. 2. Carefully close cap to PCR tube. Mix reagents by gently tapping tube bottom on lab bench. 3. Store your sample on ice or in the refrigerator until ready for amplification along with other student samples. 4. Program and start thermal cycler with a step file: (Your facilitator will help you do this!) Reaction volume is 50 µl. Hold 94 C 5 minutes C 1 minute cycles 65 C 1 minute 72 C 1 minute Holds 72 C 10 minutes for final file 4 C Hold at end of run Prepare an agarose gel while your PCR reaction is in progress. Procedure C: Preparation of 1.5% Agarose Gel 1. Your facilitator will show each group how to prepare their 1.5% agarose gels, in 1x TAE buffer. Weigh g agarose and transfer to an erlenmeyer flask. Add 40 ml 1X TAE buffer. Melt agarose in microwave or on a hot plate, swirling frequently. Let the agarose cool to the touch (but not solidify). Then add 5 µl of Ethidium Bromide stock (2 mg/ml) to agarose. Pour the agarose into a prepared gel casting tray. 5

6 NOTE FACILITATORS SHOULD RESTRICT HANDLING OF ETHIDIUM BROMIDE SINCE IT IS A HEALTH HAZARD. Procedure D. Agarose Gel Electrophoresis 1. Use permanent marker to label a clean 1.5 ml tube 2. Transfer 20 µl of your PCR sample to the labeled tube 3. Add 4 µl of loading dye to the PCR sample. Close tube and mix by tapping tube on bottom of lab bench or by pulsing in a microcentrifuge. 4. Add 20 µl of the PCR/loading dye sample into your assigned well of the 1.5% agarose gel. Expel any air in the tip before loading and be careful not to punch the tip of the pipet through the bottom of the sample well. 5. In each gel, run one lane with a DNA marker. Load 10 µl DNA marker into the gel as described above. The DNA marker contains a ladder of DNA fragments ranging in size from 100 bp to 2100 bp. Sizes are 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, etc. The 500 bp, 1000bp, and 2100 bp bands are especially bright and easy to locate. The size of your PCR product can be estimated by comparing its position to the DNA marker. 6. Electrophorese agarose gels at 150 volts for 35 minutes to one hour. Adequate separation will have occurred when the bromophenol dye front has moved at least 50 mm from wells. 7. Following electrophoresis turn off power supply, disconnect power leads, remove gel, and place on the UV transilluminator to observe and take photos of gel. Wear gloves at this step. RESULTS AND DISCUSSION 1. Examine the photograph of the stained gel containing your sample and those from other individuals. Orient the photograph with the sample wells at the top. First, look for a diffuse (fuzzy) band of primer dimer that might appear toward the bottom of the gel, at the same position in each lane. Primer dimer is not amplified human DNA, but is an artifact of the PCR reaction that results from primers amplifying themselves. Excluding primer dimer, interpret the allele bands in each lane of the gel: a. No bands visible. This usually results from an error during sample preparation, such as losing the cheek cell pellet or failing to resuspend Chelex beads prior to transferring solutions between test tubes. b. One band visible. The simplest explanation is that the individual is homozygous at the D1S80 locus, having inherited the same allele on maternal and paternal chromosome 1. However, since the samples were electrophoresed on agarose 6

7 gels, then it is more likely that the individual is, in fact, heterozygous, but the two alleles are so similar in size that they cannot be resolved (separated) in this gel system. Another possibility is that a larger allele (with many repeats) has failed to amplify efficiently. c. Two bands visible. The individual is heterozygous at the D1S80 locus. Often, the larger allele amplifies less efficiently and appears less intense than the smaller one. d. Three or more bands visible. The two brightest bands are likely the true alleles. Additional bands may occur when the primers bind nonspecifically to chromosome loci other than D1S80 and give rise to additional amplification products. 2. Population studies have identified 29 alleles at the D1S80 allele, and estimate that 90% of individuals are heterozygous at this locus. Determine the number of different alleles represented among your classmates and the percent of heterozygous individuals. How does your class data compare with that of the general population? What reasons can you give for differences? 3. Based on your results, do you think this protocol could be used to link a suspect with a crime or establish a paternity relationship? Why do you think so? How could you modify the experiment to improve its ability to positively identify individuals? 7

8 WORKING WITH YOUR DATA A. Analysis of D1S80 PCR products using agarose gel electorphoresis The figure below is an example of an agarose gel containing DNA from four students PCR reactions. In one lane there is a DNA marker; the other lanes contain PCR products for the D1S80 locus. Use the DNA marker to approximate the size, in base pairs, of the PCR products in each sample lane. For example, in the diagram below, the PCR products in lane 1 can be approximated at 640 base pairs (bp) and 320 bp. In lane 2, we see a single band at 400 bp. We can conclude from the data that the individuals in lane 1 and lane 2 are heterozygous and homozygous, respectively, with respect to the D1S80 locus. How would you interpret lanes 3 and 4? M

9 After you have determined the size of the DNA bands on the gel, you can calculate the number of repeats at the D1S80 locus. But, first, look at an example for calculating the size of the PCR product for a given allelle. An allele composed of 17 repeats is used in this example. Notice that repeat unit 1 has 14 bp and the remaining repeat units have 16bp. However, this 2 bp difference doesn t change the number of repeats found at D1S80 during analysis, therefore we will assume repeat 1 has 16 bp in the following formulas. 5 1 TCAGCCCA-AGG-AAG 2 ACAGACCACAGGCAAG 3 GAGGACCACCGGAAAG GAAGACCACCGGAAAG GAAGACCACCGGAAAG GAAGACCACAGGCAAG 7 8 GAGGACCACCGGAAAG GAGGACCACCGGCAAG GAGGACCACCAGGAAG GAGGACCACCAGGAAG 9 GAGGACCACCGGCAAG 12 GAGGACCACCGGCAAG GAGGACCACCAGGAAG GAGAACCACCAGGAAG GAGGACCACCAGGAAG 16 GAGGACCACCAGGAAG 17 GAGGACCACTGGCAAG 3 The length of D1S80 alleles can be calculated as follows: 115 bp upstream (region from "left" primer to repeat unit 1) + (total number of repeats ) x 16 base pairs + 32 bp downstream (region from last repeat to right primer) length of D1S80 in bp 5 upstream D1S80 downstream

10 In the example above, the size of the PCR products expected would be: (17 x 16) + 32 = 419 B. Calculate the number of repeats at your D1S80 loci To determine the number of repeats from the size of the bands on a gel, work backwards. Number of repeats = (# of BP estimated from Gel (115+32)) 16 Or to make it really simple Number of repeats = (# of BP estimated from Gel 147) 16 In the gel on page 8, lane 1 contains a PCR product of 640 bp and 320 bp. Thus, the number of repeats would be: (( ) / 16) = 30.81, or ~ 31 repeats. The number of repeats for 320 bp would be: (( ) / 16) = 10.81, or ~11 repeats. Calculate the number of repeats for the 400bp band: (( ) / 16) = repeats Now, calculate the number of repeats at your D1S80 loci. Fill in the box with the estimated size of your D1S80 locus and solve the equation below: (if you are heterozygous, you will have to do this twice!) Number of repeats = -147 ( ) 16 Finally, determine the size of the PCR products and the number of repeats for your DNA and the DNA of the other persons working at your table. Record the information in the table on the next page. 10

11 lane name size of DNA (base pairs) #of repeats homozygous or heterozygous Label and tape the picture of your gel in the space below. 11

Exercise 5: Detection of a Human Alu Element by PCR

Exercise 5: Detection of a Human Alu Element by PCR Exercise 5: Detection of a Human Alu Element by PCR (adapted from Dolan DNA Learning Center, Cold Spring Harbor Laboratory, NY and Science Outreach, Washington University, St. Louis, MO) Background Information

More information

LAB 16 HUMAN DNA TYPING USING POLYMERASE CHAIN REACTION (PCR)

LAB 16 HUMAN DNA TYPING USING POLYMERASE CHAIN REACTION (PCR) LAB 16 HUMAN DNA TYPING USING PCR LAB 16 HUMAN DNA TYPING USING POLYMERASE CHAIN REACTION (PCR) STUDENT GUIDE GOAL The goal of this lab is to use polymerase chain reaction (PCR) to compare a human DNA

More information

Extraction from Buccal Epithelial Cells

Extraction from Buccal Epithelial Cells Genomic DNA Extraction from Buccal Epithelial Cells The purpose of this lab is to collect a DNA sample from the cells that line the inside of your mouth and to use this sample to explore one of the most

More information

Quick Guide Lesson 1 Cheek Cell DNA Template Preparation

Quick Guide Lesson 1 Cheek Cell DNA Template Preparation Quick Guide Lesson 1 Cheek Cell DNA Template Preparation 1. Label one 1.5 ml micro test tube with your initials. Label one screwcap tube containing 200 µl of InstaGene matrix with your initials. 2. Obtain

More information

PTC DNA Fingerprint Gel

PTC DNA Fingerprint Gel BIO 141 PTC DNA Fingerprint Analysis (Modified 3/14) PTC DNA Fingerprint Gel taster non- non- non- non- 100 bp taster taster taster taster taster taster taster ladder Tt tt Tt TT tt tt Tt tt 500 bp 300

More information

HiPer RT-PCR Teaching Kit

HiPer RT-PCR Teaching Kit HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The

More information

The Techniques of Molecular Biology: Forensic DNA Fingerprinting

The Techniques of Molecular Biology: Forensic DNA Fingerprinting Revised Fall 2011 The Techniques of Molecular Biology: Forensic DNA Fingerprinting The techniques of molecular biology are used to manipulate the structure and function of molecules such as DNA and proteins

More information

Lab 5: DNA Fingerprinting

Lab 5: DNA Fingerprinting Lab 5: DNA Fingerprinting You are about to perform a procedure known as DNA fingerprinting. The data obtained may allow you to determine if the samples of DNA that you will be provided with are from the

More information

HiPer Restriction Fragment Length Polymorphism (RFLP) Teaching Kit

HiPer Restriction Fragment Length Polymorphism (RFLP) Teaching Kit HiPer Restriction Fragment Length Polymorphism (RFLP) Teaching Kit Product Code: HTBM026 Number of experiments that can be performed: 5/25 Duration of Experiment Protocol:3.5 hours Agarose Gel Electrophoresis:1

More information

electrode Electrophoresis tank with gel Agarose Gel DNA migration

electrode Electrophoresis tank with gel Agarose Gel DNA migration Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 5 - Electrophoresis & Quantification of PCR DNA Please bring a portable USB memory device to lab Once we have performed

More information

GEL ELECTROPHORESIS OF PLASMID DNA

GEL ELECTROPHORESIS OF PLASMID DNA Purpose: In this lab you will determine the size of a circular piece of bacterial DNA (a plasmid) by cutting it into smaller pieces with enzymes and finding the size of the pieces using agarose gel electrophoresis.

More information

EXPERIMENT 10: Restriction Digestion and Analysis of Lambda DNA

EXPERIMENT 10: Restriction Digestion and Analysis of Lambda DNA Biochemistry Lab CHE 431 Page 1 of 7 OBJECTIVE: EXPERIMENT 10: Restriction Digestion and Analysis of Lambda DNA DNA splicing, the cutting and linking of DNA molecules, is one of the basic tools of modern

More information

Isolation and Electrophoresis of Plasmid DNA

Isolation and Electrophoresis of Plasmid DNA Name Date Isolation and Electrophoresis of Plasmid DNA Prior to lab you should be able to: o Explain what cloning a gene accomplishes for a geneticist. o Describe what a plasmid is. o Describe the function

More information

Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR)

Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR) Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR) Goals of the lab: 1. To understand how DNA s chemical properties can be exploited for purification 2. To learn practical applications of DNA purification

More information

LAB 7 DNA RESTRICTION for CLONING

LAB 7 DNA RESTRICTION for CLONING BIOTECHNOLOGY I DNA RESTRICTION FOR CLONING LAB 7 DNA RESTRICTION for CLONING STUDENT GUIDE GOALS The goals of this lab are to provide the biotech student with experience in DNA digestion with restriction

More information

The Chinese University of Hong Kong School of Life Sciences Biochemistry Program CUGEN Ltd.

The Chinese University of Hong Kong School of Life Sciences Biochemistry Program CUGEN Ltd. The Chinese University of Hong Kong School of Life Sciences Biochemistry Program CUGEN Ltd. DNA Forensic and Agarose Gel Electrophoresis 1 OBJECTIVES Prof. Stephen K.W. Tsui, Dr. Patrick Law and Miss Fion

More information

PCR-based Alu-Human DNA Typing

PCR-based Alu-Human DNA Typing The Biotechnology Education Company Revised and Updated NOTE: PCR Cycling Conditions have changed. Please review your PCR program before performing the experiment. PCR-based Alu-Human DNA Typing Storage:

More information

Troubleshooting Sequencing Data

Troubleshooting Sequencing Data Troubleshooting Sequencing Data Troubleshooting Sequencing Data No recognizable sequence (see page 7-10) Insufficient Quantitate the DNA. Increase the amount of DNA in the sequencing reactions. See page

More information

Microbiology Laboratory: MOLECULAR IDENTIFICATION OF UNKNOWN BACTERIA

Microbiology Laboratory: MOLECULAR IDENTIFICATION OF UNKNOWN BACTERIA Microbiology Laboratory: MOLECULAR IDENTIFICATION OF UNKNOWN BACTERIA Classical Microbiology courses are typically structured to introduce the identification of bacterial species using a series of biochemical

More information

VNTR Human DNA Typing Using PCR

VNTR Human DNA Typing Using PCR Revised and Updated Edvo-Kit #334 VNTR Human DNA Typing Using PCR 334 Experiment Objective: In this experiment, students will extract their own genomic DNA. The Polymerase Chain Reaction (PCR) and Agarose

More information

Crime Scenes and Genes

Crime Scenes and Genes Glossary Agarose Biotechnology Cell Chromosome DNA (deoxyribonucleic acid) Electrophoresis Gene Micro-pipette Mutation Nucleotide Nucleus PCR (Polymerase chain reaction) Primer STR (short tandem repeats)

More information

The Effects of Plasmid on Genotype and Phenotype (Revised 1/31/96) Introduction

The Effects of Plasmid on Genotype and Phenotype (Revised 1/31/96) Introduction The Effects of Plasmid on Genotype and Phenotype (Revised 1/31/96) Introduction Plasmids are small circular DNA molecules that often found in bacteria in addition to the large circular DNA molecule of

More information

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis (DGGE) Craig Tepper 9/10 Modified from Laboratory of Microbial Ecology, University of Toledo Denaturing Gradient Gel Electrophoresis (DGGE) Background Information Denaturing gradient gel electrophoresis (DGGE)

More information

BIOTECHNOLOGY. What can we do with DNA?

BIOTECHNOLOGY. What can we do with DNA? BIOTECHNOLOGY What can we do with DNA? Biotechnology Manipulation of biological organisms or their components for research and industrial purpose Usually manipulate DNA itself How to study individual gene?

More information

Objectives: Vocabulary:

Objectives: Vocabulary: Introduction to Agarose Gel Electrophoresis: A Precursor to Cornell Institute for Biology Teacher s lab Author: Jennifer Weiser and Laura Austen Date Created: 2010 Subject: Molecular Biology and Genetics

More information

Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction

Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction Objectives Understand the process of DNA isolation Perform DNA isolation using cheek cells Use thermal cycler and Taq polymerase to perform DNA

More information

4 General PCR Methods Page

4 General PCR Methods Page Table of Contents General PCR Methods Page PCR Protocol Selection Guide...65.1 Basic PCR... 66.1.1 Hot Start PCR - The new Standard... 66.1.1.1 Reagents and Equipment Required... 66.1.1.2 General Considerations

More information

Alu-Human DNA Typing Using PCR

Alu-Human DNA Typing Using PCR Revised and Updated Edvo-Kit #333 Alu-Human DNA Typing Using PCR 333 Experiment Objective: In this experiment, students will extract their own genomic DNA. The Polymerase Chain Reaction (PCR) and Agarose

More information

1/12 Dideoxy DNA Sequencing

1/12 Dideoxy DNA Sequencing 1/12 Dideoxy DNA Sequencing Dideoxy DNA sequencing utilizes two steps: PCR (polymerase chain reaction) amplification of DNA using dideoxy nucleoside triphosphates (Figures 1 and 2)and denaturing polyacrylamide

More information

RESTRICTION ENZYME ANALYSIS OF DNA

RESTRICTION ENZYME ANALYSIS OF DNA University of Massachusetts Medical School Regional Science Resource Center SUPPORTING MATHEMATICS, SCIENCE AND TECHNOLOGY EDUCATION 222 Maple Avenue, Stoddard Building Shrewsbury, MA 01545-2732 508.856.5097

More information

LAB 11 PLASMID DNA MINIPREP

LAB 11 PLASMID DNA MINIPREP LAB 11 PLASMID DNA MINIPREP STUDENT GUIDE GOAL The objective of this lab is to perform extraction of plasmid DNA and analyze the results. OBJECTIVES After completion, the student should be able to: 1.

More information

Cloning of genes from genomic DNA: Part 3-Restriction Enzyme Digestion and Agarose Gel Electrophoresis

Cloning of genes from genomic DNA: Part 3-Restriction Enzyme Digestion and Agarose Gel Electrophoresis Cloning of genes from genomic DNA: Part 3-Restriction Enzyme Digestion and Agarose Gel Electrophoresis Continuing from our isolation of genomic DNA and PCR amplification of either the evenskipped gene

More information

HiPer Restriction Digestion Teaching Kit

HiPer Restriction Digestion Teaching Kit HiPer Restriction Digestion Teaching Kit Product Code: HTBM015 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1-1.5 hours Agarose Gel Electrophoresis : 1 hour Storage

More information

LAB 12 PLASMID MAPPING

LAB 12 PLASMID MAPPING STUDENT GUIDE LAB 12 PLASMID MAPPING GOAL The objective of this lab is to perform restriction digestion of plasmid DNA and construct a plasmid map using the results. OBJECTIVES After completion, the student

More information

STA DARD OPERATI G PROCEDURE FOR THE DETECTIO OF AFRICA SWI E FEVER VIRUS (ASFV) BY CO VE TIO AL POLYMERASE CHAI REACTIO (PCR)

STA DARD OPERATI G PROCEDURE FOR THE DETECTIO OF AFRICA SWI E FEVER VIRUS (ASFV) BY CO VE TIO AL POLYMERASE CHAI REACTIO (PCR) STA DARD OPERATI G PROCEDURE FOR THE DETECTIO OF AFRICA SWI E FEVER VIRUS (ASFV) BY CO VE TIO AL POLYMERASE CHAI REACTIO (PCR) jmvizcaino@vet.ucm.es Av/ Puerta de Hierro s/n. 28040 Madrid. Tel: (34) 913944082

More information

Genetics Faculty of Agriculture and Veterinary Medicine

Genetics Faculty of Agriculture and Veterinary Medicine Genetics 10201232 Faculty of Agriculture and Veterinary Medicine Instructor: Dr. Jihad Abdallah Topic 15:Recombinant DNA Technology 1 Recombinant DNA Technology Recombinant DNA Technology is the use of

More information

DNA Electrophoresis Lesson Plan

DNA Electrophoresis Lesson Plan DNA Electrophoresis Lesson Plan Primary Learning Outcomes: Students will learn how to properly load a well in an agarose gel. Students will learn how to analyze the results of DNA electrophoresis. Students

More information

RT-PCR: Two-Step Protocol

RT-PCR: Two-Step Protocol RT-PCR: Two-Step Protocol We will provide both one-step and two-step protocols for RT-PCR. We recommend the twostep protocol for this class. In the one-step protocol, the components of RT and PCR are mixed

More information

Biotechnology Explorer

Biotechnology Explorer Biotechnology Explorer Chromosome 16: PV92 PCR Informatics Kit Catalog #166-2100EDU explorer.bio-rad.com Note: Kit contains temperature-sensitive reagents. Open immediately upon arrival and store components

More information

PCR Optimization. Table of Contents Fall 2012

PCR Optimization. Table of Contents Fall 2012 Table of Contents Optimizing the Polymerase Chain Reaction Introduction.....1 Review of Mathematics........ 3 Solving Problems of Dilution and Concentration: Two Approaches.. 4 Experiment Overview 7 Calculations

More information

Genomic Diversity Laboratory Department of Ecology and Evolutionary Biology University of Michigan

Genomic Diversity Laboratory Department of Ecology and Evolutionary Biology University of Michigan HOW-TO MANUAL Genomic Diversity Laboratory Department of Ecology and Evolutionary Biology University of Michigan Cleaning How to clean your bench? A good practice is to clean your bench before and after

More information

Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR

Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR Edvo-Kit #345 Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR Experiment Objective: The objective of this experiment is for students to isolate human DNA and use PCR to amplify

More information

Plant Genomic DNA Extraction using CTAB

Plant Genomic DNA Extraction using CTAB Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however

More information

PicoMaxx High Fidelity PCR System

PicoMaxx High Fidelity PCR System PicoMaxx High Fidelity PCR System Instruction Manual Catalog #600420 (100 U), #600422 (500 U), and #600424 (1000 U) Revision C Research Use Only. Not for Use in Diagnostic Procedures. 600420-12 LIMITED

More information

ABSTRACT. Promega Corporation, Updated September 2008. http://www.promega.com/pubhub. 1 Campbell-Staton, S.

ABSTRACT. Promega Corporation, Updated September 2008. http://www.promega.com/pubhub. 1 Campbell-Staton, S. A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA... A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA from Shed Reptile Skin ABSTRACT

More information

GEL ELECTROPHORESIS BACKGROUND the gel. The gels we'll be making are flat and rectangular, or slab-like, in shape.

GEL ELECTROPHORESIS BACKGROUND the gel. The gels we'll be making are flat and rectangular, or slab-like, in shape. Gel Electrophoresis GEL ELECTRPRESIS BACKGRUND the gel. The gels we'll be making are flat and rectangular, or slab-like, in shape. We want you to understand how gel electrophoresis works. Electrophoresis

More information

First class: Basic skills in a molecular biology lab

First class: Basic skills in a molecular biology lab First class: Basic skills in a molecular biology lab a) Prepare digest with BamHI or BglII, for the following DNAs: FUGW, genomic DNA, FUCEdW. b) Prepare 1% agarose gel c) Precipitate genomic DNA d) Run

More information

Terra PCR Direct Polymerase Mix User Manual

Terra PCR Direct Polymerase Mix User Manual Clontech Laboratories, Inc. Terra PCR Direct Polymerase Mix User Manual Cat. Nos. 639269, 639270, 639271 PT5126-1 (031416) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain

More information

Session 5: Restriction Digestion and Making an Agarose Gel. Session 6: Analysis of Results by Gel Electrophoresis

Session 5: Restriction Digestion and Making an Agarose Gel. Session 6: Analysis of Results by Gel Electrophoresis LAB SKILLS: MOLECULAR BIOLOGY This course introduces students to basic techniques in molecular biology, through extracting their own DNA and genotyping themselves at a mtdna locus by restriction digestion.

More information

CLONING IN ESCHERICHIA COLI

CLONING IN ESCHERICHIA COLI CLONING IN ESCHERICHIA COLI Introduction: In this laboratory, you will carry out a simple cloning experiment in E. coli. Specifically, you will first create a recombinant DNA molecule by carrying out a

More information

Procedure for RNA isolation from human muscle or fat

Procedure for RNA isolation from human muscle or fat Procedure for RNA isolation from human muscle or fat Reagents, all Rnase free: 20% SDS DEPC-H2O Rnase ZAP 75% EtOH Trizol Chloroform Isopropanol 0.8M NaCitrate/1.2M NaCl TE buffer, ph 7.0 1. Homogenizer-probe

More information

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System)

More information

Human Mitochondrial Analysis using PCR and Electrophoresis

Human Mitochondrial Analysis using PCR and Electrophoresis Lab 9. Human Mitochondrial Analysis using PCR and Electrophoresis Prelab Assignment Before coming to lab, read carefully the introduction and the procedures for each part of the experiment, and then answer

More information

Appendix D: Pre-lab Assignments and Gel Electrophoresis Worksheet

Appendix D: Pre-lab Assignments and Gel Electrophoresis Worksheet Appendix D: Pre-lab Assignments and Gel Electrophoresis Worksheet PCR Pre-Lab (pg. 1-3) PCR Pre-Lab Answers (pg. 4-7) RNAi Pre-Lab (pg. 8) RNAi Pre-Lab Answers (pg. 9-10 Gel Electrophoresis Worksheet (pg.

More information

DNA CLONING: amplification of unique DNA molecules. In vivo-in different host cells

DNA CLONING: amplification of unique DNA molecules. In vivo-in different host cells DNA CLONING DNA CLONING: amplification of unique DNA molecules In vitro-pcr In vivo-in different host cells POLYMERASE CHAIN REACTION (PCR) PCR THE POLYMERASE CHAIN REACTION (PCR) PROVIDES AN EXTREMELY

More information

Troubleshooting Guide for DNA Electrophoresis

Troubleshooting Guide for DNA Electrophoresis Troubleshooting Guide for Electrophoresis. ELECTROPHORESIS Protocols and Recommendations for Electrophoresis electrophoresis problem 1 Low intensity of all or some bands 2 Smeared bands 3 Atypical banding

More information

Forensic DNA Testing Terminology

Forensic DNA Testing Terminology Forensic DNA Testing Terminology ABI 310 Genetic Analyzer a capillary electrophoresis instrument used by forensic DNA laboratories to separate short tandem repeat (STR) loci on the basis of their size.

More information

Transformation Protocol

Transformation Protocol To make Glycerol Stocks of Plasmids ** To be done in the hood and use RNase/DNase free tips** 1. In a 10 ml sterile tube add 3 ml autoclaved LB broth and 1.5 ul antibiotic (@ 100 ug/ul) or 3 ul antibiotic

More information

BacReady TM Multiplex PCR System

BacReady TM Multiplex PCR System BacReady TM Multiplex PCR System Technical Manual No. 0191 Version 10112010 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI Detailed Experimental

More information

Quantitative Telomerase Detection Kit (QTD Kit)

Quantitative Telomerase Detection Kit (QTD Kit) Quantitative Telomerase Detection Kit (QTD Kit) Catalog No. MT3010, MT3011, MT3012 For Research Use Only. Not for use in diagnostic procedures 1 Table of Contents 1. Introduction Background Product Overview

More information

Super One-step RT-PCR Kit for Virus Detection

Super One-step RT-PCR Kit for Virus Detection Super One-step RT-PCR Kit for Virus Detection For fast and sensitive one-step RT-PCR with highly specificity www.tiangen.com/en SD121221 Super One-step RT-PCR Kit Kit Contents Contents Cat. no. SD103 SD103

More information

Alu PV92 PCR. Table of Contents Fall 2012

Alu PV92 PCR. Table of Contents Fall 2012 Table of Contents Ah, Lou! There Really are Differences Between Us! Introduction..1 The Polymerase Chain Reaction.3 Laboratory Exercise: DNA Extraction..... 4 Important Laboratory Practices... 4 DNA Preparation

More information

3.5 Renibacterium salmoninarum (Bacterial Kidney Disease, BKD)

3.5 Renibacterium salmoninarum (Bacterial Kidney Disease, BKD) 3.5 Renibacterium salmoninarum (Bacterial Kidney Disease, BKD) - 1 R 3.5 Renibacterium salmoninarum (Bacterial Kidney Disease, BKD) enibacterium salmoninarum infections can occur at any life stage in salmonid

More information

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency

More information

HiPer Total RNA Extraction Teaching Kit

HiPer Total RNA Extraction Teaching Kit HiPer Total RNA Extraction Teaching Kit Product Code: HTBM012 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1 hour Agarose Gel Electrophoresis: 1 hour Storage Instructions:

More information

Experiment 2: DNA fingerprinting for microbial source tracking

Experiment 2: DNA fingerprinting for microbial source tracking Experiment 2: DNA fingerprinting for microbial source tracking Goal: To fingerprint strains of E. coli, and determine the source, as best as possible, for each of the strains. Introduction The Clean Water

More information

Lecture 38: DNA Fingerprinting

Lecture 38: DNA Fingerprinting Lecture 38: DNA Fingerprinting (DNA technology) The most awesome and powerful tool acquired by man since the splitting of atoms - The Time Magazine (USA) Conventional fingerprint of an individual comes

More information

SOP Title: Multiplex-PCR check of genomic DNA isolated from FFPE tissue for its usability in array CGH analysis

SOP Title: Multiplex-PCR check of genomic DNA isolated from FFPE tissue for its usability in array CGH analysis SOP Title: Multiplex-PCR check of genomic DNA isolated from FFPE tissue for its usability in array CGH analysis The STORE processing methods were shown to be fit-for purpose for DNA, RNA and protein extraction

More information

Agarose Gel Electrophoresis with Food Color- Teacher Guide

Agarose Gel Electrophoresis with Food Color- Teacher Guide Page 1 of 7 Project Home Gateway to the Project Laboratory Activities What the Project can do in the classroom Biotechnology Resources Favorite resources online and in print Agarose Gel Electrophoresis

More information

GENOTYPING ASSAYS AT ZIRC

GENOTYPING ASSAYS AT ZIRC GENOTYPING ASSAYS AT ZIRC A. READ THIS FIRST - DISCLAIMER Dear ZIRC user, We now provide detailed genotyping protocols for a number of zebrafish lines distributed by ZIRC. These protocols were developed

More information

Lecture 27: Agarose Gel Electrophoresis for DNA analysis

Lecture 27: Agarose Gel Electrophoresis for DNA analysis Lecture 27: Agarose Gel Electrophoresis for DNA analysis During Lecture 9 and 10 we have studied basics of protein electrophoresis. Recalling our discussion during lecture, protein needs to be boiled with

More information

How Does a Genetic Counselor Detect Mutant Genes? SECTION E. How Genes and the Environment Influence Our Health CHAPTER 3

How Does a Genetic Counselor Detect Mutant Genes? SECTION E. How Genes and the Environment Influence Our Health CHAPTER 3 CHAPTER 3 How Genes and the Environment Influence Our Health SECTION E How Does a Genetic Counselor Detect Mutant Genes? Chapter 3 Modern Genetics for All Students T 211 Chapter 3: Section E Background

More information

Worked solutions to student book questions Chapter 13 DNA

Worked solutions to student book questions Chapter 13 DNA Q1. Copy the structural formula of deoxyribose sugar. Now indicate where covalent bonds form to link this group to: a a phosphate group b a base group A1. Q2. Give the sequence of bases that will pair

More information

T C G A A C T G A G G A C T A T. 4) What type of bonds hold the two strands of DNA together? Are they strong or weak bonds?

T C G A A C T G A G G A C T A T. 4) What type of bonds hold the two strands of DNA together? Are they strong or weak bonds? Fo Sci DNA Questions #1 Name Key Use PPT slides 1-11 to answer the following questions. 1) Who are these gentlemen and what did they discover? James Watson and Frances Crick discovered the structure of

More information

E.Z.N.A. Plant Seed Direct PCR Kit

E.Z.N.A. Plant Seed Direct PCR Kit E.Z.N.A. Plant Seed Direct PCR Kit TQ2900-00 TQ2900-01 20 preps 100 preps June 2013 E.Z.N.A. Plant Seed Direct PCR Kit Table of Contents Introduction...2 Kit Contents/Storage and Stability...3 Suggested

More information

Application Guide... 2

Application Guide... 2 Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied

More information

UltraClean Soil DNA Isolation Kit

UltraClean Soil DNA Isolation Kit PAGE 1 UltraClean Soil DNA Isolation Kit Catalog # 12800-50 50 preps New improved PCR inhibitor removal solution (IRS) included Instruction Manual (New Alternative Protocol maximizes yields) Introduction

More information

Paper Clip PCR PURPOSE

Paper Clip PCR PURPOSE Paper Clip PCR PURPOSE The purpose of this activity is to reinforce basic DNA concepts and to introduce students to the technique of PCR and its many uses. Part one of the activity uses a virtual PCR animation

More information

Sequencing a Genome: Inside the Washington University Genome Sequencing Center. Activity Supplement Paper PCR (DNA Amplification)

Sequencing a Genome: Inside the Washington University Genome Sequencing Center. Activity Supplement Paper PCR (DNA Amplification) Sequencing a Genome: Inside the Washington University Genome Sequencing Center Activity Supplement (DNA Amplification) Project Outline The multimedia project Sequencing a Genome: Inside the Washington

More information

The MiniOne TM Reagent Kit: Electrophoresis 101

The MiniOne TM Reagent Kit: Electrophoresis 101 The MiniOne TM Reagent Kit: Cat#: M3001 2 3 Reagent Kit Components Other Required Materials Laboratory Safety Experiment Procedures Teacher s Guide Student Worksheet Ordering Consumables Table of Contents

More information

GenScript BloodReady TM Multiplex PCR System

GenScript BloodReady TM Multiplex PCR System GenScript BloodReady TM Multiplex PCR System Technical Manual No. 0174 Version 20040915 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI

More information

Introductory Gel Electrophoresis

Introductory Gel Electrophoresis 21-1147 Teacher Demonstration Kit 21-1148 8-Station Classroom Kit Introductory Gel Electrophoresis TEACHER S MANUAL WITH STUDENT GUIDE Teacher s Manual Overview.....................................................................

More information

Gel Electrophoresis of DNA

Gel Electrophoresis of DNA Gel Electrophoresis of DNA The explosion of molecular biology techniques that began in the mid-1970s and continues today has provided us with tools to examine the physical structure of DNA, its nucleotide

More information

Crime Scene Investigator PCR Basics Kit

Crime Scene Investigator PCR Basics Kit Biotechnology Explorer Crime Scene Investigator PCR Basics Kit Catalog #166-2600EDU explorer.bio-rad.com Note: Kit contains temperature-sensitive reagents. pen immediately upon arrival and store components

More information

Identification of the VTEC serogroups mainly associated with human infections by conventional PCR amplification of O-associated genes

Identification of the VTEC serogroups mainly associated with human infections by conventional PCR amplification of O-associated genes Identification of the VTEC serogroups mainly associated with human infections by conventional PCR amplification of O-associated genes 1. Aim and field of application The present method concerns the identification

More information

TCB No May Technical Bulletin. GS FLX and GS Junior Systems. Short Fragment Removal for the Amplicon Library Preparation Procedure

TCB No May Technical Bulletin. GS FLX and GS Junior Systems. Short Fragment Removal for the Amplicon Library Preparation Procedure TCB No. 2011-007 May 2013 Technical Bulletin GS FLX and GS Junior Systems Short Fragment Removal for the Amplicon Library Preparation Procedure Introduction Some library preparation methods may result

More information

Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability

Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability 21-1376 21-1377 21-1378 21-1379 21-1380 21-1381 Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability IMPORTANT

More information

Application Note. Biotechnology Explorer Crime Scene Investigator PCR Basics. Kit: A Real-Time PCR Extension

Application Note. Biotechnology Explorer Crime Scene Investigator PCR Basics. Kit: A Real-Time PCR Extension Biotechnology Explorer Crime Scene Investigator PCR Basics Kit: Table of Contents Introduction.............................................. 2 Learning Objectives......................................

More information

UltraClean Forensic DNA Isolation Kit (Single Prep Format)

UltraClean Forensic DNA Isolation Kit (Single Prep Format) UltraClean Forensic DNA Isolation Kit (Single Prep Format) Catalog No. Quantity 14000-10 10 preps 14000-S 1 prep Instruction Manual Please recycle Version: 10302012 1 Table of Contents Introduction...

More information

PCR Polymerase Chain Reaction

PCR Polymerase Chain Reaction Biological Sciences Initiative HHMI PCR Polymerase Chain Reaction PCR is an extremely powerful technique used to amplify any specific piece of DNA of interest. The DNA of interest is selectively amplified

More information

ELUTION OF DNA FROM AGAROSE GELS

ELUTION OF DNA FROM AGAROSE GELS ELUTION OF DNA FROM AGAROSE GELS OBTECTIVE: To isolate specific bands or regions of agarose-separated DNA for use in subsequent experiments and/or procedures. INTRODUCTION: It is sometimes necessary to

More information

RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis

RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis INTRODUCTION This laboratory will demonstrate the basics of electrophoresis and the theory behind the separation of molecules on an agarose

More information

INTRODUCTION. Detecting Genetically Modified Foods by PCR

INTRODUCTION. Detecting Genetically Modified Foods by PCR INTRODUCTION Genetic engineering is responsible for the so-called second green revolution. Genes that encode herbicide resistance, insect resistance, draught tolerance, frost tolerance, and other traits

More information

Computer 6B. Forensic DNA Fingerprinting

Computer 6B. Forensic DNA Fingerprinting Forensic DNA Fingerprinting Computer 6B Scientists working in forensic labs are often asked to perform DNA profiling or fingerprinting to analyze evidence in law enforcement, mass disasters, and paternity

More information

An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH

An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH DNA basic principles DNA carries the information of life on Earth DNA is present in all organisms (some viruses use RNA)

More information

21-1230 21-1230A 21-1231 21-1231A 21-1232 21-1232A. Using an Alu Insertion Polymorphism to Study Human Populations

21-1230 21-1230A 21-1231 21-1231A 21-1232 21-1232A. Using an Alu Insertion Polymorphism to Study Human Populations 21-1230 21-1230A 21-1231 21-1231A 21-1232 21-1232A Using an Alu Insertion Polymorphism to Study Human Populations Using an Alu Insertion Polymorphism to Study Human Populations IMPORTANT INFORMATION Storage:

More information

PicoMaxx High Fidelity PCR System

PicoMaxx High Fidelity PCR System PicoMaxx High Fidelity PCR System Instruction Manual Catalog #600420 (100 U), #600422 (500 U), and #600424 (1000 U) Revision B Research Use Only. Not for Use in Diagnostic Procedures. 600420-12 LIMITED

More information

CAP BIOINFORMATICS Su-Shing Chen CISE. 10/5/2005 Su-Shing Chen, CISE 1

CAP BIOINFORMATICS Su-Shing Chen CISE. 10/5/2005 Su-Shing Chen, CISE 1 CAP 5510-8 BIOINFORMATICS Su-Shing Chen CISE 10/5/2005 Su-Shing Chen, CISE 1 Genomic Mapping & Mapping Databases High resolution, genome-wide maps of DNA markers. Integrated maps, genome catalogs and comprehensive

More information

AGAROSE GEL ELECTROPHORESIS:

AGAROSE GEL ELECTROPHORESIS: AGAROSE GEL ELECTROPHORESIS: BEST PRACTICES (BACK TO THE BASICS) Unit of Tropical Laboratory Medicine April 2009 Marcella Mori WORKFLOW OF AGAROSE GEL ELECTROPHORESIS: THREE STEPS Agarose gel electrophoresis

More information

Project Proposal - Analysis of Historical Retroviral Contributions to The Modern Human Genome

Project Proposal - Analysis of Historical Retroviral Contributions to The Modern Human Genome Project Proposal - Analysis of Historical Retroviral Contributions to The Modern Human Genome Prepared for: The Summer Research Program, Saint Mary s College of CA School of Science Prepared by: Thomas

More information