* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 345 - Timstar
Genetic engineering wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Primary transcript wikipedia , lookup
Molecular Inversion Probe wikipedia , lookup
DNA polymerase wikipedia , lookup
Metagenomics wikipedia , lookup
Point mutation wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Genomic library wikipedia , lookup
DNA profiling wikipedia , lookup
DNA vaccination wikipedia , lookup
Non-coding DNA wikipedia , lookup
Designer baby wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
DNA supercoil wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Molecular cloning wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Genome editing wikipedia , lookup
Microevolution wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Epigenomics wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
History of genetic engineering wikipedia , lookup
Helitron (biology) wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Microsatellite wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
The Biotechnology Education Company ® EDVO-Kit # 345 Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR Storage: See Page 3 for specific storage instructions EXPERIMENT OBJECTIVE: The objective of this experiment is for students to isolate human DNA and use PCR to amplify a segment of the TAS2R38 gene, which is responsible for detecting the bitter taste of PTC. Digestion of the PCR products and analysis by agarose gel electrophoresis are used to identify the presence of SNP. Genotype is linked to phenotype by tasting the PTC paper. EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com 345.130821 2 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 xxx Table of Contents Experiment Components 3 Experiment Requirements 4 Background Information 5 Experiment Procedures Experiment Overview 11 Module I: Isolation of DNA from Human Cheek Cells 12 Module II: Amplification of the PTC Regions 13 Module III: Restriction Digest of the PTC PCR Products 14 Module IV: Separation of PCR Products and Digestion Products by Agarose Gel Electrophoresis Module V: Staining Agarose Gels 15 17 Module VI: Confirmation of Bitter Tasting Ability with PTC Paper 19 Study Questions 20 Instructor’s Guidelines Pre-Lab Preparations 21 Experiment Results and Analysis 27 Answers to Study Questions 28 Appendices 29 A Troubleshooting Guides 30 B Preparation and Handling of PCR Samples With Wax 32 C Bulk Preparation of Agarose Gels 33 Material Safety Data Sheets can be found on our website: www.edvotek.com The PCR process and Taq DNA polymerase are covered by patents owned by Hoffman-LaRoche, Inc. EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc. UltraSpec-Agarose, PCR EdvoBead, and FlashBlue are trademarks of EDVOTEK, Inc. 345.130821 EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 3 EDVO-Kit # 345 Experiment Components Component Storage A Room Temperature ❑ -20°C Freezer -20°C Freezer -20°C Freezer -20°C Freezer Room temperature -20°C Freezer -20°C Freezer -20°C Freezer Room temperature Room temperature ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ Experiment # 345 contains material for up to 25 reactions. Sample volumes are very small. It is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipetting. Spin samples for 10-20 seconds at maximum speed. B C D E F G H I • • Tubes with PCR EdvoBeads™ Each PCR EdvoBead™ contains • dNTP Mixture • Taq DNA Polymerase Buffer • Taq DNA Polymerase • MgCl2 • Reaction Buffer PTC Primer mix concentrate 100 base pair ladder Control DNA concentrate TE buffer Proteinase K Restriction Enzyme Reaction Buffer Restriction Enzyme Dilution Buffer Hae III Restriction Enzyme PTC Paper Control Taste Paper Check (√) NOTE: Components B and D are supplied in concentrate form and require dilution prior to setting up PCR reactions. Reagents & Supplies Store all components below at room temperature. Component 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. • • • • • • • • • • • • Check (√) UltraSpec-Agarose™ Electrophoresis Buffer (50x) 10x Gel Loading Solution InstaStain® Ethidium Bromide FlashBlue™ Stain Disposable plastic cups Conical Tube Microcentrifuge Tubes (with caps) Microcentrifuge Tubes (1.5 ml screw-cap tube – use for boiling) PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template) Wax beads (for waterbath option or thermal cyclers without heated lid) Salt packets EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com FAX: 202-370-1501 • email: [email protected] 345.130821 ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ 4 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 xxx Experiment Requirements • *If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three waterbaths. However, a thermal cycler assures a significantly higher rate of success. • • • • • • • • • • • • • • • • • • (NOT included in this experiment) Thermal cycler (EDVOTEK Cat. # 541 highly recommended) or three waterbaths* Waterbath (55º C and boiling) (EDVOTEK Cat. # 539 highly recommended) Horizontal gel electrophoresis apparatus D.C. power supply Balance Microcentrifuge UV Transilluminator or UV Photodocumentation system (use if staining with InstaStain® Ethidium Bromide) UV safety goggles White light visualization system (optional - use if staining with FlashBlue™) Automatic micropipets (5-50 µl) with tips Microwave or hot plate Pipet pump 250 ml flasks or beakers Hot gloves Disposable laboratory gloves Ice buckets and ice Distilled or deionized water Spring water Bleach solution 345.130821 EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 5 345 Background Information SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) C G C T A G C G 1 SNP 2 A T T C G C T A A G Figure 1: Single Nucleotide Polymorphism (SNP) occurs at a single base-pair location (C/T). SNPs may fall within any region of the genome – the coding sequences of genes, the noncoding sequences of genes, or regions in-between genes (the intergenic regions). When an SNP falls within a coding sequence, that alteration may not necessarily change the amino acid sequence of the subsequent protein produced due to degeneracy of the genetic code. On the other hand, SNPs that do not fall into a coding sequence may still affect that region of DNA in such a way by inducing gene splicing, transcription factor binding, or by altering the sequence of non-coding RNA. Variations in our DNA sequences can affect many aspects of human physiology - how we develop diseases, respond to pathogens, process chemicals, drugs , and many more. For example, sickle cell anaemia is caused by a single nucleotide mutation in the ß-globin chain of hemoglobin. This, in turn, causes the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine, thus altering the shape of the blood cells. Instead of having the usual disc-like shape that is flexible, enabling them the pass even the smallest blood vessels, sickle cells are long “sickleshaped” red blood cells that block the flow of blood through the blood vessels. Sickle cells can lead to various complications, including acute chest syndrome, organ damage, gallstone, and stroke. THE BITTER TASTE AND THE BITTER COMPOUND PHENYLTHIOCARBAMIDE (PTC) Individuals vary greatly in their sensitivity to the bitter compound Phenylthiocarbamide (PTC). This is one of the best-known genetic traits in the human population and has been a popular teaching tool for genetic inheritance. In this experiment, we examine variable regions to distinguish differences in individuals by identifying two different forms (alleles) of a gene related to one’s ability to taste PTC (Figure 2). Figure 2: The structure of PTC. The famous European geneticist Professor Ed V. Otek, tested his rather large genetics class of students for the ability to taste PTC. He discovered that this gene has two alleles: the dominant allele (T), which confers the ability to taste PTC, and the recessive non-taster allele (t). A person inherits one copy of the gene from each of his / her parents. The combination of these different alleles within an individual is referred to as a genotype, which in turn dictates the phenotype: Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Background Information A T Single Nucleotide Polymorphisms (SNPs) are DNA sequence variations that occur in the genome sequence when a single nucleotide is altered. The genetic code is specified by the four nucleotide letters: A (Adenine), C (Cytosine), T (Thymine), and G (Guanine). SNP occurs when a single nucleotide, such as a T, replaces one of the other three nucleotides A, G, or C (Figure 1). Each person has many SNPs that together create a unique DNA fingerprint profile for that individual. 6 Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 345 Background Information Background Information in this case, whether an individual is a “taster” or “non taster.” PTC-tasters have one of two possible genotypes; either they are homozygous dominant and have two copies of the taster allele (TT), or they are heterozygous and have one taster allele and one non-taster allele (Tt). “Non-tasters” are homozygous recessive and have two copies of the non-taster allele (tt). Within the general population, about 70% of the people tested can taste PTC, whereas the other 30% cannot. THE PTC SENSITIVITY GENE – TAS2R38 Studies show that there is an inherited component that effects how people taste PTC. In 2003, more than 70 years after its original discovery, the gene responsible for PTC sensitivity, TAS2R38, was identified. Analysis of SNPs within the coding region of TAS2R38 revealed that PTC taster and nontaster alleles differ in 3 amino acids. There are two common forms (or alleles) of the PTC gene: a dominant tasting allele and a recessive non-tasting allele. The shape of the receptor protein determines how strongly it can bind to PTC. Since all people have two copies of every gene, combinations of the alleles determine whether someone finds PTC intensely bitter, somewhat bitter, or tasteless. As discussed earlier, there are 3 positions within the gene that control the ability to taste PTC (as shown in table below). Table I - Relationship of Variations at Specific locations in TAS2R38 gene and Ability to Taste PCR. Nucleotide Position Change in Nucleotide (Nontaster > Taster) Change in Codon (Nontaster > Taster) Change in Amino Acid (Nontaster > Taster) 145 G > C GCA > CCA Alanine > Proline 785 T > C GTT > GCT Valine > Alanine 886 A > G ATC > GTC Valine > Isoleucine RESTRICTION ENZYME DIGESTION Restriction enzymes are endonucleases which catalyze the cleavage of the phosphate bonds within both strands of DNA. The distinguishing feature of restriction enzymes is that they only cut at very specific sequences of bases. A single base change in the recognition results in the inability of the restriction enzyme to cut the DNA at that sequence location. Differences in the sequence of DNA at that specific location can be quickly identified using restriction enzyme digestion. A restriction enzyme requires a specific double-stranded recognition sequence of nucleotide bases to cut DNA. Recognition sites are usually 4 to 8 base pairs in length. Cleavage occurs within or near the site. Recognition sites are frequently symmetrical, i.e., both DNA strands in the site have the same base sequence when read 5’ to 3’. Such sequences are called palindromes. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 7 345 Background Information Homozygous Taster (TT) Heterozygous Taster (Tt) Non-Taster (tt) Extraction of DNA from Buccal cells Extraction of DNA from Buccal cells PCR Amplification of PTC region PCR Amplification of PTC region PCR Amplification of PTC region GGCGGCCACT GGCGGGCACT GGCGGCCACT GGCGGGCACT PCR Product 221 bp PCR Product 221 bp PCR Product 221 bp Hae III Restriction Digest Hae III Restriction Digest Hae III Restriction Digest GGCGG Figure 3: Determining PTC Genotype CCACT GGCGGGCACT GGCGGGCACT GGCGG CCACT Agarose Gel Electrophoresis Agarose Gel Electrophoresis 44 bp Fragment 177 bp Fragment Agarose Gel Electrophoresis 221 bp Fragment (remains uncut) 221 bp Fragment 44 bp Fragment 177 bp Fragment Lane 1 Lane 2 Lane 3 (TT) (Tt) (tt) 221 bp Fragment 177 bp Fragment PTC TASTER: Homozygous Taster (TT) = sizes of 177 & 44 bp Heterozygous Taster (Tt) = 1 allele remains uncut at 221 bp while the other allele cuts and generates fragments of 177 bp and 44 bp. PTC NON-TASTER: Homozygous recessive (tt) = remains uncut at 221 bp 44 bp Fragment Consider the recognition site and cleavage pattern of EcoRI and HaeIII below. The cleavage positions are indicated by arrows. Digestion with EcoRI produces assymmetric “sticky ends”, whereas HaeIII restriction enzyme cleavage produces “blunt’ ends. Cleavage pattern of EcoRI: ↓ 5’-GAATTC-3’ Eco RI 3’-CTTAAG-5’ Digestion ↑ 5’-G AATTC-3’ 3’-CTTAA G-5’ Cleavage pattern of HaeIII: 5’-GG CC-3’ 3’-CC GG-5’ 5’-GG and CC-3’ 3’-CC and GG-5’ In the example of the PTC gene, Hae III only cuts the taster allele (5’-GGCGGCCACT-3’). The polymorphism present in the non- taster allele (5’-GGCGGGCACT-3’) changes a single base change in the restriction enzyme recognition site, so Hae III can not digest non-taster DNA. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Background Information Extraction of DNA from Buccal cells 8 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 Background Information Background Information THE POLYMERASE CHAIN REACTION (PCR) The PCR reaction is a DNA amplification technique that revolutionized almost all aspects of biological research. The procedure was invented by Dr. Kary Mullis while at Cetus Corporation in 1984. Dr. Mullis was awarded a Nobel Prize for his work in 1994. PCR amplification can produce millions of copies from a small quantity of DNA. The enormous utility of PCR is based on its procedural simplicity and specificity. Since the first application of PCR to diagnose sickle cell anemia, a large number of amplifications have successfully been developed. PCR has made amplification of DNA an effective alternative to cloning. It is currently routinely used in forensics, paternity/kinship testing, and the identification of human remains. In preparation for PCR amplification, a set of two DNA “primers” are designed to target a specific region of the genomic sequence containing the gene of interest. The primers are two synthetic oligonucleotides typically 15-30 base pairs in length that are synthesized so that they correspond to the start and end of a specific region of the DNA sequence to be amplified. In this experiment, the template DNA is from individuals who show differences in their ability to taste PTC. The extracted DNA is called the “template.” Freshly isolated DNA from biological sources will yield the best amplification. DNA extracted from stored specimens may be degraded and therefore less suitable for amplification. In addition to the two primers, the four deoxynucleotides (dATP, dCTP, dGTP, and dTTP) that are the precursors building blocks of DNA and a thermally stable DNA polymerase are required. The most commonly used DNA polymerase is the enzyme Taq polymerase, which is purified from the thermophilic bacterium Thermus aquaticus that inhabits hot springs. This enzyme is stable at near-boiling temperatures. The PCR process requires a sequential heating and cooling cycle of the mixture at three different temperatures. It is efficiently performed in a thermal cycler, an instrument that is programmed to rapidly heat, cool, and maintain samples at designated temperatures for varying amounts of time. In the first step of the PCR reaction the mixture is heated to near boiling (94°C),to allow the two complementary DNA strands to be denatured. This step, known as “denaturation,” disrupts the hydrogen bonds between the strands and causes the complete separation of the two DNA strands. In the second PCR step, the sample is cooled to a temperature in the range of 45°C - 65°C. In this step, known as “annealing,” the two primers, which are present in great excess to the separated DNA template strands, bind to their target complements. In the third step, known as “extension” (also called DNA synthesis), the temperature is raised to an intermediate value (usually 72°C). At this temperature the Taq polymerase is maximally functional. It adds the precursor nucleotides to the primers to complete the synthesis of the new complementary strands based on the traditional Watson-Crick base pairing. These three steps--denaturation, annealing, and extension constitute one PCR “cycle.” Each cycle doubles the amount of Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 9 345 Background Information One common problem that occurs during PCR is the production of unwanted amplification products. This may be due to contamination of the sample or nonspecific annealing (to the wrong segment). If this were to occur in an early cycle, the incorrect copy will also be amplified. To reduce contamination, autoclaved tubes and pipet tips, as well as sterile water should be used. Gloves should always be worn when performing PCR. Minimizing the concentration of the primers may curtail the production of unwanted PCR due to nonspecific annealing. Another common technique is a “hot start” step, in which the PCR reagents are introduced in the reaction only after the DNA is fully denatured at 94°C. In this experiment, the students will use the PCR-RFLP method to examine the presence of the polymorphism. Students will use the PCR to amplify the polymorphic region of the TAS2R38 gene. The amplified DNA will be digested with the restriction enzyme Hae III to determine their genotype at position 145, which correlates with the ability to taste PTC. Agarose gel electrophoresis of the restriction-digested PCR products will reveal the 2 alleles of the TAS2R38 gene, indicating whether a student is homozygous or heterozygous for the taster phenotype. In the final module, students will test their ability to taste the bitter PTC and correlate their genotype with their phenotype. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com The Experiment the target DNA. Calculated mathematically, if the cycle is repeated n times the number of copies will be an exponential enlargement of 2n. For example, ten cycles will produce 210 or 1,048,576 copies. The PCR process is typically repeated for 20-40 cycles, theoretically amplifying the target sequence to millions of copies. In practice, however, the amount of product often reaches a maximum after about 35 cycles. This is due to the depletion of reaction components and loss of Taq polymerase activity. The exact temperature and incubation time required for each of the three steps depends upon several factors, including the length of the DNA target and the Guanine /Cytosine (GC) content of the primer/ target. 10 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 Background Information Target Sequence = Separation of two DNA strands 5' 3' 3' 5' 5' 3' 3' 5' Cycle 1 = Primer 2 5' 5' 3' Cycle 2 3' 3' 3' 5' Denature 94°C 3' 5' Anneal 2 primers 40°C - 65°C 5' 5' 3' 5' 5' 5' 3' Cycle 3 The Experiment = Primer 1 5' 3' 5' 5' 5' 5' 5' 5' 5' 3' 3' 5' 5' 3' 5' 5' 3' 5' 5' 5' 3' Extension 72°C 3' 5' 5' 3' 5' 5' 3' 3' 5' 5' 3' 5' 5' Figure 4 The Polymerase Chain Reaction (PCR) - Three Cycles Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 11 345 Experiment Overview and General Instructions EXPERIMENT OBJECTIVE Wear gloves and safety goggles MODULE I: 50 min. IMPORTANT Isolation of DNA from Cheek cells Be sure to READ and UNDERSTAND the instructions completely BEFORE starting the experiment. If you are unsure of something, ASK YOUR INSTRUCTOR! MODULE II: 80 min. • Wear gloves and goggles while working in the laboratory. • Exercise caution when working in the laboratory – you will be using equipment that can be dangerous if used incorrectly. • Wear protective gloves when working with hot reagents like boiling water and melted agarose. • DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS. • Always wash hands thoroughly with soap and water after working in the laboratory. • Contaminated laboratory waste (saliva solution, cup, pipet, etc.) must be disinfected with 15% bleach solution prior to disposal. Be sure to properly dispose any biological samples according to your institutional guidelines. Amplification of the PTC Regions MODULE III: 40 min. Restriction Digest of the PTC PCR Product MODULE IV: 50-70 min. Separation of PCR Products and Digestion Products by Agarose Gel Electrophoresis MODULE V: 5 min. Staining Agarose Gels MODULE VI: 10 min. Confirmation of Bitter Tasting Ability with PTC Paper Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com The Experiment The objective of this experiment is for students to isolate human DNA and use PCR to amplify a segment of the TAS2R38 gene, which is responsible for detecting the bitter taste of PTC. Digestion of the PCR products and analysis by agarose gel electrophoresis are used to identify the presence of SNP. Genotype is linked to phenotype by tasting the PTC paper. 12 Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 345 Module I: Isolation of DNA from Human Cheek Cells 1.5 ml 2. 1. 4. SPIN 3. T.C. T.C. 60 T.C. Full speed 2 min. The Experiment sec. 5. Swirl 7. 6. 55° C Vortex or Flick 8. 99° C 15 15 140 µl Lysis Buffer min. min. 99 99 Warning! Students should use screw-cap tubes when boiling samples. Vigorously 20 sec. © 2013 Edvotek® All Rights Reserved. 9. SPIN 10. 80 µl Supernatant Low speed 2 min. 1. 2. LABEL a 1.5 ml screw top microcentrifuge tube and a cup with your lab group and/or initials. RINSE your mouth vigorously for 60 seconds using 10 ml saline solution. EXPEL the solution into cup. STEP 4: 3. SWIRL the cup gently to resuspend the cells. TRANSFER 1.5 ml of solution into If cell pellet size is not large enough, repeat the labeled tube. steps 3 - 4 until you 4. CENTRIFUGE the cell suspension for 2 min. at full speed to pellet the cells. have a large size POUR off the supernatant, but DO NOT DISTURB THE CELL PELLET! Repeat pellet. For best results, steps 3 and 4 twice more. make sure your cell pellet is at least the 5. RESUSPEND the cheek cells in 140 µl lysis buffer by pipetting up and down or size of a match head. by vortexing vigorously. 6. CAP the tube and PLACE in a waterbath float. INCUBATE the sample in a 55° C waterbath for 15 min. STEP 7: If a vortex is not available, mix 7. MIX the sample by vortexing or flicking the tube vigorously for 20 seconds. samples by flicking the 8. INCUBATE the sample in a 99° C waterbath for 15 min. Be sure to use screwtube vigorously for 20 cap tubes when boiling DNA isolation samples. seconds. 9. CENTRIFUGE the cellular lysate for 2 minutes at low speed (6000 rpm). 10. TRANSFER 80 µl of the supernatant to a clean, labeled microcentrifuge tube. PLACE tube in ice. 11. PROCEED to Module II: Amplification of the TAS2R38 Locus. OPTIONAL STOPPING POINT: The extracted DNA may be stored at -20°C for amplification at a later time. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 13 345 Module II: Amplification of the PTC Regions 2. 1. • 20 µl PTC primer • 5 µl extracted DNA • PCR EdvoBead™ Gently mix 3. SPIN #1 4. 5. If your thermal cycler does not have a heated lid, it is necessary to overlay the PCR reaction with wax to prevent evaporation. See Appendix B for guidelines. 1. ADD 20 µL PTC primer mix, 5 µl extracted DNA (or Control DNA) and one PCR EdvoBead™ to a labeled 0.2 ml or 0.5 ml PCR tube (depending on the Thermal Cycler). 2. MIX the PCR sample. Make sure the PCR EdvoBead™ is completely dissolved. 3. CENTRIFUGE to collect the sample at the bottom of the tube. 4. AMPLIFY DNA using PCR: PCR cycling conditions: Initial denaturation 94°C for 4 minutes 94° C for 30 seconds 64° C for 45 seconds 35 cycles 72° C for 45 seconds Final Extension 72° C for 5 minutes 5. PLACE tubes on ice. PROCEED to Module III: Restriction Digest of PCR Product. OPTIONAL STOPPING POINT: The PCR samples may be stored at -20° C for restriction digest at a later time. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com The Experiment NOTES AND REMINDERS: 14 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 Module III: Restriction Digest of the PTC Product 2. The Experiment 1. PCR amplified DNA (20µl) #1 Reaction buffer (5µl) Hae III Restriction Enzyme (5µl) 4. SPIN #1 37° C 30 99 3. Gently mix. min. 5. 6. Gel Loading Dye (5µl) #1 Gel Loading Dye (5µl) Water (15µl) Digested DNA #2 Uncut DNA © 2013 Edvotek® All Rights Reserved. 1. ADD 20 µl PCR amplified DNA, 5 µl Reaction Buffer (F), and 5 µl Restriction Enzyme Hae III to a 1.5 ml microcentrifuge tube. Label this tube “1”. Save the remaining 5 µl uncut PCR sample to set up as a control later. Label this as tube “2”. 2. Gently MIX the restriction digest (tube “1”) by gently tapping the tube. 3. Quickly CENTRIFUGE to collect sample at the bottom of the tube. 4. INCUBATE the digest for 30 min at 37°C. 5. ADD 5 µl 10X Gel Loading Dye to 25 µl restriction-digested DNA. 6. ADD 5 µl 10X Gel Loading Dye and 15 µl ultrapure water to 5 µl uncut DNA (tube “2” saved from step 1) as a control. PROCEED to Module IV: Agarose Gel Electorphoresis. OPTIONAL STOPPING POINT: The restriction digests may be stored at -20°C for electrophoresis at a later time. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 15 345 Module IV: Separation of Digestion Products by Agarose Gel Electrophoresis 1. 50 2. 3. x Concentrated buffer Distilled water 1:00 IMPORTANT: In this experiment, students will perform Agarose Gel Electrophoresis twice: Agarose Flask 4. 5. • Once for HaeIII Digested PCR Products (requiring one 7 x 14 cm gel per group). 60°C 6. WAIT Pour 60°C 1. 2. 3. 4. 5. 6. 7. • Once for Undigested PCR Products (requiring one 7 x 7 cm gel per group). Each gel can be shared by 4 students. Place wellformer templates (combs) in the first set of notches. 7. 20 If you are unfamiliar with agarose gel prep and electrophoresis, detailed instructions and helpful resources are available at www.edvotek.com min. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A). MIX agarose powder with 1X buffer in a 250 ml flask (see Table B, page 16). DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by Wear gloves swirling the flask. Continue to HEAT the solution in 15-second bursts until the and safety goggles agarose is completely dissolved (the solution should be clear like water). COOL agarose to 60° C with careful swirling to promote even dissipation of heat. While agarose is cooling, SEAL the ends of the gelcasting tray with the rubber end caps. PLACE the well Table template (comb) in the appropriate notch. 1x Electrophoresis Buffer (Chamber Buffer) POUR the cooled agarose solution into the prepared Dilution Total Volume EDVOTEK Distilled gel-casting tray. The gel should thoroughly solidify 50x Conc. Required Model # + Water Buffer within 20 minutes. The gel will stiffen and become 6 ml 294 ml 300 ml M6+ less transparent as it solidifies. REMOVE end caps and comb. Take particular care 8 ml 392 ml 400 ml M12 when removing the comb to prevent damage to the 20 ml 980 ml 1000 ml M36 wells. A Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com The Experiment Caution! Flask will be HOT! 16 Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 345 Module IV: Separation of Digestion Products by Agarose Gel Electrophoresis 8. 9. Reminder: Pour Before loading the samples, make sure the gel is properly oriented in the apparatus chamber. The Experiment 1X Diluted Buffer 11. 10. Wear gloves and safety goggles 8. PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer (See Table B for recommended volumes). The gel should be completely submerged. 9. LOAD the entire sample (30 µL) into the well. RECORD the position of the samples in Table 1, below. 10. PLACE safety cover. CHECK that the gel is properly oriented. Remember, the DNA samples will migrate toward the positive (red) electrode. 11. CONNECT leads to the power source and PERFORM electrophoresis (See Table C for time and voltage guidelines). 12. After electrophoresis is complete, REMOVE the gel and casting tray from the electrophoresis chamber and proceed to STAINING the agarose gel. Table B Individual 2.0% UltraSpec-Agarose™ Gel Size of Gel Casting tray 1X Dilluted Buffer 7 x 7 cm 25 ml 7 x 14 cm Table C 50 ml + Amt of Agarose Table 1 Gel 1: PCR Reaction for PTC Gene Lane 0.50 g 1.0 g Time and Voltage Guidelines (2.0% Agarose Gels) 1 100 bp ladder 2 Undigested PCR Product - Control 3 Undigested PCR Product - Taster 1 4 Undigested PCR Product - Taster 2 5 Undigested PCR Product - Taster 3 6 Undigested PCR Product - Taster 4 Gel 2: RFLP Analysis to Detect PTC Polymorphisms Time: 7 x 7 cm gel Time: 7 x 14 cm gel Volts ~4.0 cm migration ~6.5 cm migration 1 100 bp ladder 125 30 min. 60 min. 2 HaeIII Digested PCR Product - Control 70 60 min. 120 min. 3 HaeIII Digested PCR Product - Taster 1 50 90 min. 150 min. 4 HaeIII Digested PCR Product - Taster 2 5 HaeIII Digested PCR Product - Taster 3 6 HaeIII Digested PCR Product - Taster 4 Lane Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 17 345 Module V-A: Staining Agarose Gels using InstaStain® Ethidium Bromide Preferred Method 1. 2. 3. Moisten the gel id e . U.S 5. d in Pen ent Pat g 6. - InstaStain® m Bro STAIN 300 nm 5 min. Ethid nt Pending U.S. Pate m Bromide InstaStain® Ethidiu U.S. Patent Pending WEAR GLOVES AND GOGGLES WHEN USING THIS PRODUCT. 1. Carefully REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel off of the casting tray on to a piece of plastic wrap on a flat surface. DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS. 2. MOISTEN the gel with a few drops of electrophoresis buffer. 3. Wearing gloves, REMOVE and DISCARD the clear plastic protective sheet from the unprinted side of the InstaStain® card(s). PLACE the unprinted side of the InstaStain® Ethidium Bromide card(s) on the gel. You will need 2 cards to stain a 7 x 14 cm gel. 4. With a gloved hand, REMOVE air bubbles between the card and the gel by firmly running your fingers over the entire surface. Otherwise, those regions will not stain. 5. PLACE the casting tray on top of the gel/card stack. PLACE a small weight (i.e. an empty glass beaker) on top of the casting tray. This ensures that the InstaStain® Ethidium Bromide card is in direct contact with the gel surface. STAIN the gel for 3-5 min. for an 0.8% gel or 8-10 min. for a gel 1.0% or greater. 6. REMOVE the InstaStain® Ethidium Bromide card(s). VISUALIZE the gel using a long wavelength ultraviolet transilluminator (300 nm). DNA should appear as bright orange bands on a dark background. BE SURE TO WEAR UV-PROTECTIVE EYEWEAR! Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com The Experiment 4. m idiu Eth in® Sta sta In 18 Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 345 Module V-B: Staining Agarose Gels using FlashBlue™ 1. 2. 10 x Concentrated FlashBlue™ Stain Wear gloves and safety goggles Distilled water The Experiment Flask 3. 5. 4. STAIN Pour 5 min. (-) 1 2 3 4 5 6 DESTAIN Pour 20 min. (+) 1. DILUTE 10 ml of 10x concentrated FlashBlue™ with 90 mL of water in a flask and MIX well. 2. REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel off of the casting tray into a small, clean gel-staining tray. 3. COVER the gel with the 1x FlashBlue™ stain solution. STAIN the gel for 5 minutes. For best results, use an orbital shaker to gently agitate the gel while staining. STAINING THE GEL FOR LONGER THAN 5 MINUTES WILL REQUIRE EXTRA DESTAINING TIME. 4. TRANSFER the gel to a second small tray. COVER the gel with water. DESTAIN for at least 20 minutes with gentle shaking (longer periods will yield better results). Frequent changes of the water will accelerate destaining. 5. REMOVE the gel from the destaining liquid. VISUALIZE results using a white light visualization system. DNA will appear as dark blue bands on a light blue background. Alternate Protocol: 1. 2. 3. DILUTE one mL of concentrated FlashBlue™ stain with 149 mL dH2O. COVER the gel with diluted FlashBlue™ stain. SOAK the gel in the staining liquid for at least three hours. For best results, stain gels overnight. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 19 345 Module VI: Confirmation of Bitter Tasting Ability with PTC Paper For this module, each student should receive the following materials: • PTC Paper • Control Taste Paper PROCEDURE: TASTE the Control strip of paper first. 2. TASTE the PTC strip of paper. 3. Compare the taste of the Control and the PTC paper. • Notice what the PTC paper tastes like compared to the Control paper: intensly bitter, somewhat bitter, or tasteless. • If you are a taster, the PTC paper strip will be bitter. Non-tasters will not notice a difference between either strip of paper. ANALYZE THE RESULTS: 1. Verify the outcome of your bitter tasting ability using the PTC paper with your genotype in the PCR-RFLP analysis in previous module. 2. Are you a homozygous bitter taster, a heterozygous bitter taster, or a nontaster? Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com The Experiment 1. 20 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 Study Questions The Experiment Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. How is PCR used to determine human genetics and identify polymorphisms in DNA? 2. What are the three steps in a PCR cycle and what does each step accomplish? 3. Based on what you have learned about the genotype of TAS2R38 and its phenotype, fill in the table below: Genotype Phenotype # of DNA bands predicted TT Tt tt 4. Based on your results, what is your genotype? Why? What is your phenotype? Why? How about your lab partners? 5. Do the control and PTC paper tasting results correlate with the DNA digest findings in your ability to taste? How about your lab partner? 6. Enter your classroom data in a table as shown below: Phenotype Genotype Strong Taster Weak taster Nontaster TT (homozygous) Tt (heterozygous) tt (homozygous) 7. Considering that not everyone who can taste PTC tastes it the same way, what does this tell you about classical dominant/recessive inheritance? Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 21 345 Instructor’s Guide OVERVIEW OF INSTRUCTOR’S PRELAB PREPARATION: This section outlines the recommended prelab preparations and approximate time requirement to complete each prelab activity. ADVANCE PREPARATION: Preparation For: Module I: Isolation of DNA from Cheek Cells Module II: Amplification of the PTC Regions Module III: Restriction Digest of the PTC PCR Products What to do: When: Time Required: Prepare and aliquot various reagents (Saline, Lysis buffer) Up to one day before performing the experiment. IMPORTANT: Prepare the Lysis buffer no more than one hour before performing the experiment. 30 min. Equilibrate waterbaths at 55° C and boiling. One hour before performing the experiment. 5 min. Prepare and aliquot various reagents (Primer, DNA template, ladder, etc.) One day to 30 min. before performing the experiment. 30 min. Program Thermal Cycler One hour before performing the experiment. 15 min. Prepare and aliquot various reagents (Reaction buffer, HaeIII Restriction enzyme, etc.) Equilibrate waterbath at 37° C One day to 30 min. before performing the experiment. IMPORTANT: Prepare the diluted HaeIII Restriction Enzyme no more than one hour before performing the experiment. 30 min. One hour before performing the experiment. 15 min. Up to one day before performing the experiment. 45 min. Module IV: Separation of PCR Products & Digestion Products by Electrophoresis Prepare diluted TAE buffer Module V: Staining Agarose Gels Prepare staining components The class period or overnight after the class period. 10 min. Module VI: Confirmation of Bitter Tasting Ability Distribute PTC taste strips and Control taste strips The class period 5 min. Prepare molten agarose and pour gel EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com FAX: 202-370-1501 • email: [email protected] 345.130821 22 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 Pre-Lab Preparations: Module I Instructor’s Guide ISOLATION OF DNA FROM HUMAN CHEEK CELLS FOR MODULE I Each Student should receive: • One cup containing 10 ml of saline solution • One screw-cap tube • One microcentrifuge tube Reagents to be Shared by Two Students: • 300 µl Lysis buffer • 15% bleach solution Preparation of Saline Solution 1. To prepare the saline solution, dissolve all 8 salt packets in 500 ml of drinking water. Cap and invert bottle to mix. 2. Aliquot 10 ml of saline solution per cup. Distribute one cup per student. Preparation of Lysis Buffer (Prepared no more than one hour before starting the experiment.) 1. Add 100 µl of TE buffer (E) to the tube of Proteinase K (F) and allow the sample to hydrate for several minutes. After the sample is hydrated, pipet up and down several times to thoroughly mix the material. 2. Transfer the entire amount of the rehydrated Proteinase K solution to a 15 ml conical tube containing an additional 4 ml of TE buffer (E). 3. Invert the tube several times to mix. Label this tube “Lysis Buffer”. 4. Aliquot 300 µl of Lysis Buffer into 13 labeled microcentrifuge tubes. 5. Distribute one tube of “Lysis Buffer” to each student pair. Warning !! Remind students to only use screw-cap tubes when boiling their DNA samples. The snap-top tubes can potentially pop open and cause injury. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 23 345 Pre-Lab Preparations: Module II Preparation of the PTC Primer Reagents to be Shared by Two Students: • 50 µl PTC Primer mix 1. Thaw the PTC Primer Mix Concentrate (B) on ice. 2. Add 1 ml of TE Buffer (E) to the tube of Primer Mix Concentrate. Cap tube and mix. 3. Label 13 microcentrifuge tubes “PTC Primer”. Aliquot 50 µl of the diluted Primer Mix into the 13 microcentrifuge tubes. Place the tubes on ice until they are needed. 4. Distribute one tube of diluted PTC Primer to each student pair. Preparation of the Control DNA 1. This kit includes enough DNA to set up 4 control reactions. At least one control reaction should be performed per class to confirm that PCR was successful. 2. Thaw the tube of Control DNA Concentrate (D) on ice. 3. Add 20 µl of TE buffer (E) to the tube containing Control DNA Concentrate. Pipet up and down to mix. 4. Dispense 8 µl of the diluted control DNA for each control reaction. Preparation of Additional Materials 1. Dispense 30 µl of 10x Gel Loading Solution for each student pair. Programming the Thermal Cycler The Thermal cycler should be programmed as outlined in Module II in the Student’s Experimental Procedure. • Accurate temperatures and cycle times are critical. A pre-run for one cycle (takes approximately 3 to 5 min) is recommended to check that the thermal cycler is properly programmed. • For thermal cyclers that do not have a heated lid, it is necessary to place a layer of wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix B for instructions. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Instructor’s Guide FOR MODULE II Each Student should receive: • One PCR tube and PCR EdvoBead™ • 30 µl Gel Loading Solution 24 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 Pre-Lab Preparations: Module III Instructor’s Guide MODULE III: RESTRICTION DIGEST OF THE PTC PRODUCT FOR MODULE III Reagents to be Shared by Two Students: • 12 µl HaeIII Restriction Enzyme • 15 µl Restriction Enzyme Reaction Buffer • 20 µl 10X Gel Loadion Solution Dilution of HaeIII restriction enzyme 1. Add 150 µl of Restriction Enzyme Dilution Buffer (H) to the tube containing the concentrated HaeIII restriction enzyme. 2. Mix the tube for 30 seconds (vortex or tap bottom of the tube) and set on ice 1 minute. 3. Dispense 12 µl of the HaeIII Restriction Enzyme for 13 tubes. Label these tubes “HaeIII” 4. Place the tubes on ice until they are needed. Each tube will be shared by a student pair. Also aliquot the following reagents: 1. Dispense 15 µl of Restriction Enzyme Reaction Buffer (G) per student pair. 2. Dispense 20 µl of 10X Gel Loading Solution per student pair. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 25 345 Pre-Lab Preparations: Module IV If students are unfamiliar with using micropipets, we recommended performing Cat. #S-44, Micropipetting Basics or Cat. #S-43, DNA DuraGel™ prior to conducting this advanced level experiment. FOR MODULE IV Each Student Group should receive: • 50x concentrated buffer • Distilled Water • UltraSpec-Agarose™ • Tube of 100 bp ladder (65 µl per 2 gels shared by 4-5 students per gel.) SEPARATION OF PCR PRODUCTS AND DIGESTION PRODUCTS BY AGAROSE GEL ELECTROPHORESIS This experiment requires two 2.0% agarose gel per student group. Each group of 4-5 students will share one 7 x 7 cm and one 7 x 14 cm gel. • A 7 x 7 cm gel is recommended for separating undigested PCR Products. • A 7 x 10 cm or 7 x 14 cm gel is recommended for separating Hae III digest of PCR Products. You can choose whether to prepare the gels in advance or have the students prepare their own. Allow approximately 30-40 minutes for this procedure. Individual Gel Preparation: Each student group can be responsible for casting their own individual gel prior to conducting the experiment. See Module III in the Student’s Experimental Procedure. Students will need 50x concentrated buffer, distilled water and agarose powder. Batch Gel Preparation: To save time, a larger quantity of agarose solution can be prepared for sharing by the class. See Appendix C. Preparing Gels in Advance: Gels may be prepared ahead and stored for later use. Solidified gels can be store under buffer in the refrigerator for up to 2 weeks. Do not freeze gels at -20ºC as freezing will destroy the gels. Gels that have been removed from their trays for storage should be “anchored” back to the tray with a few drops of molten agarose before being placed into the tray. This will prevent the gels from sliding around in the trays and the chambers. Additional Materials: • Each set of two 2.0% gel should be loaded with the 100 base pair ladder and samples from 4 or 5 students. The control PCR reaction can also be loaded in one of the wells. • Aliquot 65 µl of the 100 base-pair ladder (C) into labeled microcentrifuge tubes. • Distribute one tube of ladder per two 2.0% agarose gels shared by 4-5 students as outlined above. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Instructor’s Guide NOTE: Accurate pipetting is critical for maximizing successful experiment results. EDVOTEK Series 300 experiments are designed for students who have had previous experience with micropipetting techniques and agarose gel electrophoresis. 26 EDVO-Kit # Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR 345 Instructor’s Guide Pre-Lab Preparations: Module V FOR MODULE IV Each Student Group should receive: • 2 InstaStain® cards per 7 x 14 cm gel Wear gloves and safety goggles STAINING WITH INSTASTAIN® ETHIDIUM BROMIDE Preferred Method InstaStain® Ethidium Bromide provides the sensitivity of ethidium bromide while minimizing the volume of liquid waste generated by staining and destaining a gel. An agarose gel stained with InstaStain® Ethidium Bromide is ready for visualization in as little as 3 minutes! Each InstaStain® card will stain 49 cm2 of gel (7 x 7 cm). You will need 2 cards to stain a 7 x 14 cm gel. Use a mid-range ultraviolet transilluminator (Cat. #558) to visualize gels stained with InstaStain® Ethidium Bromide. BE SURE TO WEAR UV-PROTECTIVE EYEWEAR! • Standard DNA markers should be visible after staining even if other DNA samples are faint or absent. If bands appear faint, repeat staining with a fresh InstaStain card for an additional 3-5 min. If markers are not visible, troubleshoot for problems with electrophoretic separation. • Ethidium bromide is a listed mutagen. Wear gloves and protective eyewear when using this product. UV protective eyewear is required for visualization with a UV transilluminator. • InstaStain® Ethidium Bromide cards and stained gels should be discarded using institutional guidelines for solid chemical waste. STAINING WITH FLASHBLUE™ FlashBlue™ can be used as an alternative to Ethidium Bromide in this experiment. However, FlashBlue™ is less sensitive than InstaStain® Ethidium Bromide and will take a longer time to obtain results. FlashBlue™ stain, however, is optimized to shorten the time required for both staining and destaining steps. Agarose gels can be stained with diluted FlashBlue™ for 5 minutes and destained for only 20 minutes. For the best results, leave the gel in liquid overnight. This will allow the stained gel to “equilibrate” in the destaining solution, resulting in dark blue DNA bands contrasting against a uniformly light blue background. A white light box (Cat. #552) is recommended for visualizing gels stained with FlashBlue™. • Stained gels may be stored in destaining liquid for several weeks with refrigeration, although the bands may fade with time. If this happens, re-stain the gel. • Destained gels can be discarded in solid waste disposal. Destaining solutions can be disposed of down the drain. PHOTODOCUMENTATION OF DNA (OPTIONAL) Once gels are stained, you may wish to photograph your results. There are many different photodocumentation systems available, including digital systems that are interfaced directly with computers. Specific instructions will vary depending upon the type of photodocumentation system you are using. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 27 345 Experiment Results and Analysis Gel 1 – PCR Reaction for PTC gene PCR Products resulted from amplification of the PTC regions showed that, without the use of restriction enzymes, DNA from different individuals containing an identical region of a chromosome appear to be similar because no differences in the restriction fragment lengths are observed. Gel 2: RFLP Analysis to Detect PTC Polymorphisms Lane 1 100 bp ladder 2 HaeIII digest of PCR Product - Control 3 HaeIII digest of PCR Product - Taster 1 (example) 4 HaeIII digest of PCR Product - Taster 2 (example) 5 HaeIII digest of PCR Product - Taster 3 (example) 6 HaeIII digest of PCR Product - Taster 4 (example) • HaeIII digest of PCR Product Taster 1 (example) shows that Taster 1 is a TT Homozygous Taster. Since HaeIII restriction enzyme digests the sequence, there are two bands (a bright one at 177 bp and a fainter one at 44 bp) • HaeIII digest of PCR Product Taster 2 (example) shows that this taster is a Tt Heterozygous Taster: 3 bands (3 fragments at 221 bp, 177 bp and 44 bp) This is because half of the taster’s DNA has the taster allele, and the other half is made up of the nontaster allele. • HaeIII digests of PCR Product Tasters 3 & 4 (examples) show that they are non-tasters. Because HaeIII restriction enzyme cannot digest the non-taster sequence, PCR product will remain intact and results in one band at 221 bp. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com Instructor’s Guide Lane 1 100 bp ladder 2 Undigested PCR Product - Control 3 Undigested PCR Product - Taster 1 4 Undigested PCR Product - Taster 2 5 Undigested PCR Product - Taster 3 6 Undigested PCR Product - Taster 4 Please refer to the kit insert for the Answers to Study Questions Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR EDVO-Kit # 345 Appendices A EDVOTEK® Troubleshooting Guide B Preparation and Handling of PCR Samples With Wax C Bulk Preparation of Agarose Gels Material Safety Data Sheets: Now available for your convenient download on www.edvotek.com. EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com FAX: 202-370-1501 • email: [email protected] 345.130821 29 30 Appendix A EDVO-Kit # 345 EDVOTEK® Troubleshooting Guides DNA EXTRACTION PROBLEM: There is no cell pellet after centrifuging the cheek cell suspension. Poor DNA Extraction The extracted DNA is very cloudy. CAUSE: ANSWER: Not enough cheek cells in suspension Mouth must be vigorously rinsed for at least 60 sec. to harvest loose cheek cells. Sample not centrifuged fast enough Spin cells at maximum speed (17,000 x g) for 2 min. If your centrifuge does not reach this speed, spin at highest available speed for 4 min. Samples not mixed well enough during extraction In addition to flicking the tube, vortex or pipet up and down to mix the sample. Proteinase K inactive because it was prepared too far in advance. Prepare Proteinase K within one hour of use. Water baths not at proper temperature Use a thermometer to confirm water bath set point. Not enough DNA Try cheek cell extraction. Final DNA concentrations are usually higher. Cellular debris from pellet transferred to tube Centrifuge sample again and move supernatant to a fresh tube. Take care to avoid pellet. Cellular debris not separated from supernatant Centrifuge sample again. If possible, centrifuge at a higher speed. Move cleared supernatant to a fresh tube. RESTRICTION ENZYME DIGESTION PROBLEM: CAUSE: ANSWER: Impure DNA – some contaminants (EDTA, salts) might partially or completely inhibit activity of Hae III restriction enzyme Poor DNA extraction. Extract new DNA. Cheek cell extraction usually results in higher DNA yield. Improper dilution of enzyme Ensure that Hae III restriction enzyme was correctly diluted. Improper addition of enzyme Ensure that correct amount of Hae III restriction enzyme was added to the restriction digest. Incorrect incubation temperature Use a thermometer to confirm water bath temperature and adjust, if necessary. Unexpected Cleavage Pattern DNA sample is contaminated Prepare a new DNA sample. Smearing of digested DNA on gel Nuclease contamination Care should be taken to avoid cross contamination when setting up reactions. Undigested or incompletely digested DNA Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., 345.130821 Use fresh electrophoresis buffer and appropriate all rights reserved. Agarose running conditions voltage. The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com 345.130821 Appendix A 31 EDVO-Kit # 345 EDVOTEK® Troubleshooting Guides PCR AND ELECTROPHORESIS PROBLEM: CAUSE: ANSWER: Make sure the heated lid reaches the appropriate temperature. There is very little liquid left in tube after PCR Sample has evaporated If your thermal cycler does not have a heated lid, overlay the PCR reaction with wax (see Appendix B for details) Make sure students close the lid of the PCR tube properly. Pipetting error Make sure students pipet 20 µL primer mix and 5 µL extracted DNA into the 0.2 mL tube. Ensure that the electrophoresis buffer was correctly diluted. The gel was not prepared properly. Gels of higher concentration (> 0.8%) require special attention when melting the agarose. Make sure that the solution is completely clear of “clumps” and glassy granules before pouring gels. The gel was not stained properly. Repeat staining. Malfunctioning electrophoresis unit or power source. Contact the manufacturer of the electrophoresis unit or power source. The gel was not stained for a sufficient period of time. Repeat staining protocol. The ladder, control DNA, and student PCR products are not visible on the gel. After staining the gel, the DNA bands are faint. After staining, the ladder is visible but no PCR products are present. Repeat PCR with fresh PCR EdvoBeads™ and primers. PCR amplification was unsuccessful. Ensure that the thermal cycler has been properly programmed. See Module II for guidelines Student DNA sample was not concentrated enough. Poor DNA extraction. Extract new DNA. Cheek cell extraction usually results in higher DNA yield. Student DNA sample was degraded If DNA is not used immediately following extraction, store sample at -20°C. Wrong volumes of DNA and primer added to PCR reaction Practice using pipettes Some students have more or less amplification than others. Concentration of DNA varies by sample. There is an inherent variability in the extraction process. For best results, use cheek cell extraction. Low molecular weight band in PCR samples Primer dimer Low concentration of extracted DNA in PCR reaction. DNA bands were not resolved. Tracking dye should migrate at least 3.5 cm (if using a 7x7 cm tray), and at least 6 cm (if using a 7x14 cm tray) from the wells to ensure adequate separation. Be sure to run the gel at least 6 cm before staining and visualizing the DNA (approximately one hour at 125 V). DNA bands fade when gels are kept at 4°C. DNA stained with FlashBlue™ may fade with time Re-stain the gel with FlashBlue™ After staining, the ladder and control PCR products are visible on gel, but some student samples are not present. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com 345.130821 32 EDVO-Kit # Appendix B 345 Preparation and Handling of PCR Samples With Wax ONLY For Thermal Cyclers WITHOUT Heated Lids, or Manual PCR Using Three Waterbaths Using a wax overlay on reaction components prevents evaporation during the PCR process. HOW TO PREPARE A WAX OVERLAY 1. Add PCR components to the 0.2 ml PCR Tube as outlined in Module III. 2. Centrifuge at full speed for five seconds to collect sample at bottom of the tube. 3. Using clean forceps, add one wax bead to the PCR tube. 4. Place samples in PCR machine and proceed with Module III. PREPARING PCR SAMPLES FOR ELECTROPHORESIS 1. After PCR is completed, melt the wax overlay by heating the sample at 94° C for three minutes or until the wax melts. 2. Using a clean pipet, remove as much overlay wax as possible. 3. Allow the remaining wax to solidify. 4. Use a pipet tip to puncture the thin layer of remaining wax. Using a fresh pipet tip, remove the PCR product and transfer to a new tube. 5. Add 5 µL of 10x Gel Loading Buffer to the sample. Proceed to Module IV to perform electrophoresis. Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com 33 Appendix C EDVO-Kit # 345 Bulk Preparation of Agarose Gels To save time, electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class. Unused diluted buffer can be used at a later time and solidified agarose gel can be remelted. BULK ELECTROPHORESIS BUFFER Bulk preparation of 1X electrophoresis buffer is outlined in Table D. BATCH AGAROSE GELS (2.0%) Table D Bulk Preparation of Electrophoresis Buffer 50x Conc. Buffer + 60 ml Distilled Water Total Volume Required 2,940 ml 3000 ml (3 L) Bulk preparation of 2.0% agarose gel is outlined in Table E. Note: The UltraSpec-Agarose™ kit component is usually labeled with the amount it contains. Please read the label carefully. If the amount of agarose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount. 1. Use a 500 ml flask to prepare the diluted gel buffer. 2. Pour the appropriate amount of UltraSpec-Agarose™ into the prepared buffer. Swirl to disperse clumps. 3. With a marking pen, indicate the level of solution volume on the outside of the flask. 4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution. 5. Cool the agarose solution to 60°C with swirling to promote even dissipation of heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step 3. 6. Dispense the required volume of cooled agarose solution for casting each gel. The volume required is dependent upon the size of the gel bed. 7. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. Proceed with electrophoresis (Module IV) or store the gels at 4ºC under buffer. 60˚C Table E Batch Prep of 2.0% UltraSpec-Agarose™ Amt of Agarose 8.0 g Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2013 EDVOTEK, Inc., all rights reserved. 345.130821 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com + Diluted Buffer (1x) 400 ml