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EDVO-Kit #

337
Drosophila Genotyping Using PCR
Storage: See Page 3 for specific storage instructions

EXPERIMENT OBJECTIVE:
The objective of this experiment is to introduce students to Drosophila genotyping using the Polymerase Chain Reaction.

This experiment is designed for DNA staining with InstaStain® Ethidium Bromide.

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EVT 2012_04_26AM

The Biotechnology Education Company® • 1-800-EDVOTEK • www. Inc.com 2 EVT 2012_04_26AM . and InstaStain are registered trademarks of EDVOTEK. UltraSpec-Agarose is a trademark of EDVOTEK.0% Agarose Gel Preparation 1. The Biotechnology Education Company.Quantity Preparations Staining and Visualization of DNA with InstaStain® Ethidium Bromide Cards InstaStain® Blue: One Step Staining and Destaining Material Safety Data Sheets 33 34 32 26 28 29 30 31 18 21 23 24 9 10 11 12 14 16 3 4 5 EDVOTEK.0% Agarose Gels .337 Experiment Drosophila Genotyping Using PCR Table of Contents Experiment Components Experiment Requirements Background Information Experiment Procedures Experiment Overview and General Instructions Laboratory Safety Module I: Isolation of Drosophila DNA Module II: PCR Amplification Module III: Agarose Gel Electrophoresis Study Questions Instructor's Guidelines Notes to the Instructor Pre-Lab Preparations Experiment Results and Analysis Study Questions and Answers Appendices A B C D E F G PCR Experimental Success Guidelines Polymerase Chain Reaction Using Three Waterbaths Preparation and Handling of PCR Samples With Wax 1.edvotek. Inc.

nor administered to or consumed by humans or animals. E. D.Drosophila Genotyping Using PCR 337 Experiment Experiment Components Storage This experiment is designed for 10 lab groups. For liquid samples. EDVOTEK . H -20°C Freezer -20°C Freezer -20°C Freezer Room temperature Room temperature Room temperature Room temperature Reagents & Supplies (Store all components below at room temperature) *Drosophila must be requested two weeks in advance.com FAX: 202.800. None of the experiment components are derived from human sources.2 ml .2 ml template) Calibrated transfer pipets Wax beads (for waterbath option or thermal cyclers without heated lid) All components are intended for educational research only.1501 • email: info@edvotek. • • • • • • • • • • Wild and White Drosophila* UltraSpec-Agarose™ Electrophoresis Buffer (50x) 10x Gel Loading Solution InstaStain® Ethidium Bromide InstaStain® Blue Microcentrifuge Tubes PCR tubes (0. Spin samples for 10-20 seconds at maximum speed. A. Tubes with PCR reaction pellets™ Each PCR reaction pellet™ contains • dNTP Mixture • Taq DNA Polymerase Buffer • Taq DNA Polymerase • MgCl2 Primer mix 200 base pair ladder Control DNA TE buffer Proteinase K Potassium Acetate DNA extraction buffer Room Temperature Sample volumes are very small.EDVOTEK • www.370. G. it is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting.for thermal cyclers with 0. F. B.com EVT 2012_04_26AM 3 .edvotek. They are not to be used for diagnostic or drug purposes. THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.The Biotechnology Education Company® 1. C.

The Biotechnology Education Company® • 1-800-EDVOTEK • www. However. hot plate or burner Pipet pump 250 ml flasks or beakers Hot gloves Disposable vinyl or latex laboratory gloves Ice buckets and ice Distilled or deionized water Isopropanol *If you do not have a thermal cycler.C. using three waterbaths. power supply Balance Microcentrifuge Water bath (70° C) UV Transilluminator or UV Photodocumentation system UV safety goggles Automatic micropipets (5-50 µl) with tips Microwave. with proper care. a thermal cycler assures a significantly higher rate of success.com 4 EVT 2012_04_26AM .337 Experiment Drosophila Genotyping Using PCR Requirements • • • • • • • • • • • • • • • • • Thermal cycler (EDVOTEK Cat. PCR experiments can be conducted.edvotek. # 541 highly recommended) or three waterbaths* Horizontal gel electrophoresis apparatus D.

Drosophila eye color is determined by enzymes encoded by genes that produce pigments. This document.. The white gene encodes an enzyme that transports pigment precursors (used by subsequent enzymes) in both the pterididine and ommochrome pathways. is located at position 3B6. pteridines and ommochromes. DNA is replicated but does not segregate into daughter cells and the newly formed strands remain attached. Within one day. The white-1 mutation results from an insertion of a 4. The first three molts give rise to larval forms while the last molt results in an inactive pupal stage. an estimated 14. This RNA is then reverse transcribed. has been completely sequenced. Another advantage of Drosophila is their short life cycle. 4. In this experiment. Five different genes for eye color exist on the Drosophila X chromosome. As a result. The mobility of retrotransposons is believed to occur through an RNA intermediate. may be observed on polytene chromosomes. to double stranded DNA. 2. Polytene chromosomes. The Doc retrotransposon is a mobile genetic element that uses an RNA intermediate. by an enzyme called reverse transcriptase. is permitted for classroom/laboratory use only. the insects molt 1. such as gene encoding of proteins. Embryos of Drosophila develop in 24 hours. Two types of eye pigments. Drosophila melanogaster (the fruit fly) has been a valuable research tool in genetics and developmental biology. Drosophila was one of the first animals used to illustrate principles of modern genetics. This results in chromosomes that contain hundreds of copies attached in perfect register. This DNA intermediate can then be integrated into relatively random sites within the Drosophila genome. or any part. the gene studied in this experiment. and developmental gene regulation. all rights reserved. the fly completes its metamorphosis and emerges from the pupa as a winged adult. Background Information Duplication of this document. The sequence and mutant availabilities allow one to correlate changes at the DNA level with changes in phenotype. and several thousand genetic mutants are available for study. This duplicated state is called polytene. its entire genome. Inc. EVT 2012_04_26AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. one generation is complete within two weeks. Another laboratory advantage for studying Drosophila is the ease of visualization of nuclear material.000 genes. facilitating studies of genetic crossing. combine to impart the dull red eye color of the fruit fly. This results in neither the pteridine nor ommochrome pigments to be synthesized. After 5 days. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.165 million bases. localized expansions. divide the Drosophila genome into 102 numbered bands. the adults are fertile and may mate to begin a new life cycle. Thus the original copy of the retrotransposon is maintained. in conjunction with use of accompanying reagents. Drosophila chromosome puffs have been used in research on chromatin structure. but a new copy results at another location in the genomic DNA. transcription of the gene is repressed and no mRNA is synthesized and no transport of the enzyme occurs. and 6 days after hatching. The final result is that the eye of the mutant insect is a bleach-white color. Copyright © 2002-2012 EDVOTEK. known as “puffs”.edvotek. located on the X chromosome.4 kb mobile genetic element. Puffs unfold chromatin due to RNA synthesis and thus may be used to demarcate active genes. transcription.Drosophila Genotyping Using PCR Experiment 337 Drosophila melanogaster For almost a century. In addition to the banding system. that make up the phenotype of the gene.com 5 . the eye color of fruit flies will be studied. Using this banding. that are further subdivided into letter bands of A-F. known as white. Drosophila is inexpensive to maintain. Thus. Inc. known as the Doc retrotransposon. The letter bands are further subdivided into 1-13 numbered divisions. when stained. Salivary gland cells do not divide in adulthood. into the promoter region of the white gene.

Inc. Therefore. Furthermore. it is located on the X chromosome and is therefore also a sexlinked mutation. was awarded a Nobel Prize in 1994. and (like other retrotransposons) are believed to encode their own reverse transcriptase. The enormous utility of the PCR method is based on its ease of use and its ability to allow the amplification of small DNA fragments. female white flies must carry two copies of the mutant allele. while males carry only one copy (as males only have one X chromosome and one Y chromosome). the polymerase chain reaction (PCR) is often used. it is a recessive mutation. Although some retrotransposons can occasionally excise from their site of integration. Similar to other sex-linked recessive mutations. in conjunction with use of accompanying reagents. Inc. Since the white-1 mutation results in the suppression of the enzyme that transports pigment precursors for the ommochrome and pteridine pathways. PCR amplification (Figure 3. page 8) uses Taq DNA polymerase.. PCR. most always retain an original copy of the element as well as the newly integrated copy. or any part. This document. Duplication of this document. This enzyme is purified from a bacterium isolated from hot springs and is stable at very high temperatures. the number of LINEs continually amplify so that there are now many thousand copies of LINEs in many animals. Matings between white females and Figure 2: F2 Phenotype 2 red male (wild) 2 white female white males can only produce white offspring of both sexes (Figure 1). The white-1 Doc insertion is probably unable to excise from its location but has been shown to encode a reverse transcriptase. while those that are older are probably fixed at one locus. To detect genetic differences at the DNA level (rather than the phenotype level). has gained such widespread use that its inventor. are all about 5 kb in length.com . is permitted for classroom/laboratory use only. crossing white females with wild-type males will yield female offspring that all have wild-type eyes and males that are all white-eyed (Figure 2). Kary Mullis. Copyright © 2002-2012 EDVOTEK. On the other hand. all rights reserved. LINEs that have recently duplicated probably retain the ability to transpose to new locations.337 Drosophila Genotyping Using PCR Experiment Drosophila melanogaster XwY Xw Y XwY XwY Background Information XwXw Xw Xw XwXw XwXw Figure 1: F1 Phenotype 2 white male 2 white female XY X XwXw Xw Xw XwX XwX Y XwY XwY The Doc retrotransposon is a member of a group of retrotransposons known as long interspersed nuclear elements (LINEs). invented in 1984. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. LINEs are found in many different organisms. and is therefore probably able to replicate horizontally (to another genome location) as well as vertically (generation to generation). EVT 2012_04_26AM 6 The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.

EVT 2012_04_26AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.Drosophila Genotyping Using PCR Experiment 337 Drosophila melanogaster In the first step of PCR. while the white-1 flies will show no PCR product. the wild-type allele will show a PCR product of 220 bp. Copyright © 2002-2012 EDVOTEK. an unrelated locus (related to a wing protein) is amplified. In the second step.6 kb product in the white flies. Inc. while the polymerase remains stable. also known as the “target”. These three steps constitute one “cycle”. PCR is performed in a thermal cycler. This document.. in conjunction with use of accompanying reagents. In the third step. Inc. the sample is cooled to a temperature from 42° C to 65° C to allow hybridization of small (15-30 nucleotide) synthetic oligonucleotides.edvotek. PCR will be used to detect the presence of the Doc insertion in white mutant flies. In this experiment. which is programmed to heat the sample at the designated temperature for each of the three steps.com 7 . is permitted for classroom/laboratory use only. large PCR products are difficult to amplify and are not usually seen under normal PCR conditions. known as annealing. Therefore. However. all rights reserved. Since the white-1 allele differs from the wild-type allele by the insertion of the 4. known as denaturation. known as “primers”. the use of primers that span the insertion site can differentiate between the wild-type and white-1 alleles. to the DNA to be amplified. This process is typically repeated from 25-30 cycles. while the mutant flies will show only the 1000 bp product. the wild type flies will display both the 220 bp and 1000 bp PCR products. The primers were chosen to yield a 220 base pair product for the wild-type allele. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. As a positive control. and a 4. the temperature is raised to 72° C and the polymerase then adds nucleotides to the primers to complete each new complementary strand of the target. this PCR product is 1000 bp. Thus. or any part. the DNA complementary strands are separated at 94 degrees. In this experiment the target is the white gene locus. Background Information Duplication of this document. known as extension. amplifying the target exponentially (Figure 3).4 kb Doc element.

Copyright © 2002-2012 EDVOTEK..com . all rights reserved. is permitted for classroom/laboratory use only.337 Drosophila Genotyping Using PCR Experiment Drosophila Melanogaster Target Sequence 5' 3' 3' 5' = Separation of two DNA strands = Primer 1 = Primer 2 5' 3' Experiment Procedure Denature 94°C 3' 5' Cycle 1 5' 5' 3' 5' 3' Anneal 2 primers 40°C . Inc. EVT 2012_04_26AM 8 The Biotechnology Education Company® • 1-800-EDVOTEK • www. This document.65°C 5' 3' 5' 5' 5' 5' 3' Extension 72°C Cycle 2 5' 3' 3' 5' 5' 5' 5' 5' 5' 3' 5' 3' 5' 3' 5' 5' 3' 5' 5' 5' 5' 5' 3' 5' 5' 3' 5' 3' 5' 3' 5' 3' Cycle 3 5' 3' 5' 3' 3' 5' Figure 3: Polymerase Chain Reaction Duplication of this document. Inc. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. in conjunction with use of accompanying reagents. or any part.edvotek.

Inc. or any part.com 9 . If you will be using three waterbaths to conduct PCR. Copyright © 2002-2012 EDVOTEK. BRIEF DESCRIPTION OF EXPERIMENT: In this experiment.Drosophila Genotyping Using PCR Experiment 337 Experiment Overview and General Instructions BEFORE YOU START THE EXPERIMENT 1. EXPERIMENT OBJECTIVE: The objective of this experiment is to introduce students to Drosophila genotyping using the Polymerase Chain Reaction. EVT 2012_04_26AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. This document.4 kb Doc element. This experiment has three modules: I. also read the Appendix entitled "Preparation and Handling PCR Samples with Wax ". Write a hypothesis that reflects the experiment and predict experimental outcomes. this PCR product is 1000 bp. PCR products of the larger size are not usually seen under normal conditions. As a positive control. The primers were chosen to yield a 220 base pair product for the wild-type allele. PCR Amplification III. an unrelated locus (related to a wing protein) is amplified. If you will be conducting PCR using a thermal cycler without a heated lid. However. Therefore. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. while the mutant flies will show only the 1000 bp product. in conjunction with use of accompanying reagents. Read all instructions before starting the experiment.6 kb product in the white flies. and a 4. Thus. while the white-1 flies will show no PCR product. the use of primers that span the insertion site can differentiate between the wild-type and white-1 alleles. the wild-type allele will show a PCR product of 220 bp. PCR is used to detect the presence of the Doc insertion in white mutant flies. the wild type flies will display both the 220 bp and 1000 bp PCR products.. Experiment Procedure 3.edvotek. is permitted for classroom/laboratory use only. Inc. all rights reserved. Since the white-1 allele differs from the wild-type allele by the insertion of the 4. however. 2. Isolation of Drosophila DNA II.0% Duplication of this document. read the two appendices entitled "Polymerase Chain Reaction Using Three Waterbaths" and "Handling samples with wax overlays". Separation of PCR Reactions by Agarose Gel Electrophoresis GEL SPECIFICATIONS This experiment requires a gel with the following specifications: • • • • Recommended gel size Number of sample wells required Placement of well-former template Gel concentration required 7 x 14 cm (long tray) 4 first set of notches 1.

Copyright © 2002-2012 EDVOTEK. However. DO NOT MOUTH PIPET REAGENTS . • Although electrical current from the power source is automatically disrupted when the cover is removed from the apparatus.com . EVT 2012_04_26AM 10 The Biotechnology Education Company® • 1-800-EDVOTEK • www. Do not use the apparatus. Turn off power and unplug the equipment when not in use. Inc. first turn off the power. • 5. or any part. 2. This document.337 Drosophila Genotyping Using PCR Experiment Laboratory Safety 1. in conjunction with use of accompanying reagents.. all rights reserved.edvotek. 3. 6. Gloves and goggles should be worn routinely as good laboratory practice. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory. then unplug the power source before disconnecting the leads and removing the cover. Duplication of this document.USE PIPET PUMPS. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents. Experiment Procedure Wear gloves and safety goggles 4. Inc. in the unlikely event that a leak develops in any electrophoresis apparatus you are using. Exercise caution when using any electrical equipment in the laboratory. EDVOTEK injection-molded electrophoresis units do not have glued junctions that can develop potential leaks. is permitted for classroom/laboratory use only. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. IMMEDIATELY SHUT OFF POWER.

edvotek. WARNING: The DNA pellet is very small. Place the tube on ice. After centrifugation. This document. Use a marker to draw a circle at the bottoms of the tubes so you can locate the DNA pellet after centrifugation (see drawing at left).5 ml microcentrifuge tube. being careful not to disturb the pellet. Discard the supernatant and allow the pellet to completely dry. 3. or any part. Expel the remaining buffer and mix the solution by pipetting up and down. 8. Inc. Add 14 µl of potassium acetate to each tube and mix for 5 seconds. Use a clean pipet tip and draw up 100 µl of the DNA extraction buffer (containing Proteinase K). Resuspend the DNA pellet in 25 µl of TE buffer. Spin the tube(s) at maximum speed for 2 minutes. mark location of the pellet WARNING! Use only screwcap tubes when incubating in the waterbath for DNA isolation. insert the pipet tip into the tube containing the fly and mash the fly with the tip for 10 seconds (a small amount of buffer will release from the tip). or freeze the tubes for future use. A very small DNA pellet should be visible at the bottom of the tube where it was marked in the previous step. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. repeat steps 1-3 with a "white type" fly.com 11 . Store the tubes on ice until you are ready to proceed with Module II. Without dispensing the 100 µl of buffer into the tube. 7. Wash the pellet carefully with 20 µl of 70% ETOH. Carefully transfer just the supernatant into a fresh. 10. 5. Carefully remove and discard all of the supernatant (avoid losing the DNA pellet). Spin the tubes in a microcentrifuge at maximum speed for 5 minutes.. 2. NOTE: Dead flies should also work for the experiment. Spin at maximum speed for 5 minutes.Drosophila Genotyping Using PCR Experiment 337 Module I: Isolation of Drosophila DNA 1. (Discard the tube with the pellet after the supernatant is saved). Positive PCR results are not compromised even if flies are dead. Place the tube in the freezer for a minute to anesthetize the fly. Transfer one "wild type" fly to a 0. 13. Place the tube on ice. 6. in conjunction with use of accompanying reagents. EVT 2012_04_26AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. is permitted for classroom/laboratory use only. Duplication of this document. Protocol Hint: Use compressed air to quickly dry the DNA pellet. Precipitate the DNA in the supernatant by adding 45 µl of room temperature isopropanol to each tube (wild and white). OPTIONAL STOPPING POINT The supernatant may be stored at -20°C until the experiment is continued. Leave on ice for 5 minutes. Inc. Float both tubes in a 70°C waterbath for 15 minutes. Take care not to lose it! 11. Using a clean tube and tip. Do not use snap-top tubes. labeled 0. Copyright © 2002-2012 EDVOTEK. 12.5 ml microcentrifuge tube labeled "wild". Experiment Procedure 4. 9. all rights reserved.

2 ml) for your thermal cycler. Inc. Label one of the tubes "wild" and the other "white".337 Drosophila Genotyping Using PCR Experiment Module II: PCR Amplification PCR REACTION: 1. "wild".. If your thermal cycler is equipped with a heated lid. each containing one PCR reaction pellet™. For liquid samples. Experiment Procedure 3. it is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting.0 µl The PCR reaction pellet™ contains Taq DNA polymerase. Sample volumes are very small. Mg+2 and buffer. 6. 0. There are also materials to perform two control sample PCR reactions. in conjunction with use of accompanying reagents. 4. add the following to the correct tube making sure that the liquid comes into contact and dissolves the PCR reaction pellet™: Primer solution "white".edvotek. If your thermal cycler does not have a heated lid. 2. or any part. Spin samples for 10-20 seconds at maximum speed. This document. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. EVT 2012_04_26AM 12 The Biotechnology Education Company® • 1-800-EDVOTEK • www. or if you are cycling manually with three water baths. add one wax bead to the tube before proceeding to polymerase chain reaction cycling. or "control DNA" 5. the four deoxytriphosphates. Transfer the PCR bead to the appropriate sized tube (e.g. with your initials. Inc. Gently mix the individual PCR reaction tubes (separately) and quick spin in a microcentrifuge to collect all the sample at the bottom of the tube. proceed directly to polymerase chain reaction cycling. Copyright © 2002-2012 EDVOTEK.com . 20 µl 5. all rights reserved. Duplication of this document. is permitted for classroom/laboratory use only. Label two tubes. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube.5 ml or 0. To prepare the PCR reaction mix tube.

in conjunction with use of accompanying reagents. or any part. EVT 2012_04_26AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. Copyright © 2002-2012 EDVOTEK. 9.0% agarose gel (7 x 14 cm) and separating the PCR products by electrophoresis. Experiment Procedure NOTE: The thermal cycler can be programmed to hold the samples at 4°C overnight. OPTIONAL STOPPING POINT Samples can be frozen after addition of 5 μl of 10x Gel Loading Solution until ready for electrophoresis. Alternatively. Inc.edvotek. Proceed to instructions for preparing a 1.com 13 . may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. samples can be frozen for electrophoresis at a later time. Duplication of this document. is permitted for classroom/laboratory use only.Drosophila Genotyping Using PCR Experiment 337 Module II: PCR Amplification POLYMERASE CHAIN REACTION CYCLING 8. Add 5 µl of 10x Gel Loading Solution to the sample and store on ice until ready for electrophoresis. Inc.. This document. 10. 72°C for 90 seconds Final Extension 72°C for 5 min. 35 cycles @ 94°C for 45 seconds 67°C for 45 seconds. Put the PCR tubes in a thermal cycler programmed as follows: Initial Denaturation 94°C for 2 min. all rights reserved.

may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK.com . 5. centered and level on the platform. in conjunction with use of accompanying reagents. After the gel is cooled to 60°C: 7.337 Drosophila Genotyping Using PCR Experiment Module III: Separation of PCR Products by Agarose Gel Electrophoresis AGAROSE GEL REQUIREMENTS • Recommended gel size: 7 x 14 cm If you are unfamiliar with agarose gel preparation and electrophoresis. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape. indicate the level of the solution volume on the outside of the flask. Place the gel (on its bed) into the electrophoresis chamber. Inc. is permitted for classroom/laboratory use only. Swirl the mixture to disperse clumps of agarose powder. Place the bed on a level surface and pour the cooled agarose solution into the bed. Place a well-former template (comb) in the first set of notches at the end of the bed. If you see “crystal” particles. 10. If detectable evaporation has occurred. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer (refer to Table B on the instruction Appendix provided by your instructor). To a 250 ml flask or beaker. add agarose powder and buffer as indicated in the Reference Tables (Appendix A) provided by your instructor. 11. Inc. the agarose is not completely dissolved. After the gel is solidified. Allow the gel to completely solidify. 2. 4. 6. • • Placement of well-former template: Agarose gel concentration: first set of notches 1. 3. Heat the mixture using a microwave oven or burner to dissolve the agarose powder. Check the solution carefully.0% Experiment Procedure PREPARING THE AGAROSE GEL 1. It will become firm and cool to the touch after approximately 20 minutes. Cool the agarose solution to 60°C with careful swirling to promote even dissipation of heat.edvotek. Copyright © 2002-2012 EDVOTEK. This document. 8. EVT 2012_04_26AM 14 The Biotechnology Education Company® • 1-800-EDVOTEK • www.. or any part. add distilled water to bring the solution up to the original volume marked in step 4. 9. Make sure the comb sits firmly and evenly across the bed. Each gel can be shared by several students or groups. With a marking pen. be careful not to damage or tear the wells while removing the rubber dams or tape and comb(s) from the gel bed. Duplication of this document. IMPORTANT NOTE: Continue heating until the final solution appears clear (like water) without any undissolved particles.com 7 x 14 cm gels are recommended to achieve better resolution of the PCR products. properly oriented. all rights reserved. detailed instructions and helpful resources are available at www.edvotek.

or any part. Black Sample wells – LOADING DNA SAMPLES 1. This document. Your instructor will provide instructions for DNA staining with InstaStain® Ethidium Bromide. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor.edvotek. Reminder: Before loading the samples.you should see bubbles forming on the two electrodes. properly orient the cover and carefully snap it onto the electrode terminals..Drosophila Genotyping Using PCR Experiment 337 Module III: Separation of PCR Products by Agarose Gel Electrophoresis BEFORE LOADING THE SAMPLES This experiment requires a 1.com 15 . Copyright © 2002-2012 EDVOTEK. 5. disconnect the power and remove the gel from the bed for staining. Inc. agarose gels require staining to visualize the separated DNA samples. (Optional Step) Heat the 200 bp DNA ladder and PCR samples for two minutes at 50° C. Allow the samples to cool for a few minutes. 7. RUNNING THE GEL 4. may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK. Insert the plugs of the black and red leads into the corresponding inputs of the power source. Lane Red + Experiment Procedure 2. is permitted for classroom/laboratory use only. 6. all rights reserved. Check to see that current is flowing properly . 200 bp DNA ladder Control DNA PCR Product White DNA PCR Product Wild DNA PCR Product Record the position of your sample in the gel for easy identification after staining. STAINING AND VISUALIZATION OF DNA After electrophoresis. 8.0% agarose gel and is designed for staining with InstaStain® Ethidium Bromide. Duplication of this document. 1 2 3 4 3. Load the entire volume (30 µl) of the samples in the following sequence. Inc. EVT 2012_04_26AM The Biotechnology Education Company® • 1-800-EDVOTEK • www. in conjunction with use of accompanying reagents. Make sure the gel is completely submerged under buffer before loading the samples. After the electrophoresis is completed. make sure the gel is properly oriented in the apparatus chamber. After the DNA samples are loaded.

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