Materials Provided with the Agilent DNA Fish ID Ensemble
Contents
1. 29 2 30 Procedures Troubleshooting Guide The following table lists troubleshooting suggestions for possible scenarios you may encounter Observation The PCR reaction did not amplify a product The PCR reaction amplified multiple products If you see a note that the sum of the fragment lengths is greater than the expected amplicon size but you do not believe the sample contains multiple fish species Suggestion The extraction protocol may not have yielded any intact DNA Analyze a sample of the DNA on a spectrophotometer If no DNA is detected repeat the extraction with a piece of fish tissue between 100 and 400 mg For some samples a smaller amount of tissue yields better results If all products are around 490 bp 10 the fish sample may consist of multiple fish species The RFLP Decoder can identify multiple species using the Mixture score method If the Bioanalyzer gel of the PCR reactions shows signs of DNA degradation smeared bands the DNA sample or PCR reactions may have become contaminated Ensure the DNA is being stored at 20 C When setting up the PCR reactions use separate tips for each pipetting step If the note only appears for only one of the restriction enzymes it is unlikely that the sample is a mixture of species If the note appears for two enzymes the sample could be a mixture If all three enzymes have a note the same is very likely a mixture The sample may be contaminat2. Kit components contain DMSO Because the dye binds to nucleic acids it should be treated as a potential mutagen and used with appropriate care Wear hand and eye protection and follow good laboratory practices when preparing and handling reagents and samples Handle the DMSO stock solutions with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues Preparing the Gel Dye Mix 1 Allow DNA dye concentrate blue capped tube and DNA gel matrix red capped tube to equilibrate to room temperature for 30 min 2 Vortex DNA dye concentrate blue and add 25 ul of the dye toa 25pl dye DNA gel matrix vial red gel dye mix 3 Vortex solution well and spin down Transfer to spin filter Centrifuge at 2240 g 20 for 15 min Protect solution from light Store at 4 C Loading the Gel Dye Mix 1 Allow the gel dye mix equilibrate to room temperature for 30 min before use Put a new DNA chip on the chip priming station Pipette 9 0 ul of gel dye mix in the well marked Ja Make sure that the plunger is positioned at 1 ml and then close the chip priming station Press plunger until it is held by the clip Wait for exactly 60 s then release clip Wait for 5 s Slowly pull back plunger to 1 ml position o ol Open the chip priming station and pipette 9 0 ul of gel dye mix in the wells marked G 46 DNA Fish ID Protocol Appendix B Agilent DNA 1000 Kit Quick Start Guide 5 Lo
3. select the appropriate assay from the Assay menu Assay gt Electrophoresis gt dsDNA gt DNA 1000 Series II xsy 6 Accept the current File Prefix or modify it Data will be saved CAUTION automatically to a file with a name using the prefix you entered At this time you can also customize the file storage location Destination and Data Acquisition Parameters f Default C lent 2100 bioanalyzer2100 experthDatas2010 03 18 4 C Custom File Prefix 2100 expert max 16 characters Run sample a ee 7 Click the Start button to start the chip run After each run immediately remove the used chip from the Bioanalyzer and dispose of it according to good laboratory practice Then perform the electrode cleaning procedure described in Appendix C Bioanalyzer Electrode Cleaning Procedure on page 49 2 DNA Fish ID Protocol An image of a Bioanlyzer gel with PCR samples from 4 different fish species is shown in Figure 2 The expected results are summarized in Table 3 19 2 Procedures Figure 2 Bioanalyzer gel image of PCR reaction samples Each gel lane is labeled with the common name of the fish sample Ne amp amp g Ra S K SKF E g 1500 e e a 850 700 1 500 i d 400 300 200 150 100 S SS 15 c c MM o L 1 2 3 4 Table 3 Expected Sizes of PCR Products Sample Expected Product Size Positive c
4. 1 5 ml tube Avoid transferring any undigested material or any oily material To extract genomic DNA 1 Add 500 ul of Nucleic Acid Binding Buffer to each sample Vortex or pipet the sample repeatedly until homogenized Transfer each sample to a DNA Binding Spin Cup seated within a 2 ml receptacle tube provided and snap the tube caps onto the cups 3 Spin the samples in a microcentrifuge for 1 minute at 14 000 x g 4 For each sample discard the filtrate and replace the spin cup in the receptacle tube then add 500 ul of 1x High Salt Wash Buffer and cap the tube Spin the samples at 14 000 x g for 1 minute For each sample discard the filtrate and replace the spin cup in the receptacle tube then add 500 ul of 80 ethanol and cap the tube 7 Spin the samples at 14 000 x g for 1 minute 8 Repeat steps 6 and 7 two more times for a total of 3 washes in 80 ethanol After the third wash discard the filtrates and replace the spin cups in their receptacle tubes Spin the samples for 2 minutes at 14 000 x g to dry the fiber matrix 10 Transfer the spin cups to 1 5 ml collection tubes Add 100 ul of Elution Buffer to each cup directly on the fiber matrix Snap the tube caps onto the cups and incubate at room temperature for 1 minute 11 Spin the samples at maximum speed for 1 minute The DNA is in the collection tube DNA Fish ID Protocol Quick Reference Protocols 3 To set up and run the PCR reactions 1 Prepare
5. Nlalll 2 Prepare the reagent mixture below for the Ddel digestions and distribute 2 5 ul to the individual reaction tubes labeled for Ddel Component Volume per reaction Nuclease Free Water 1 5 ul 10x Ddel Buffer 0 5 ul 10x Ddel enzyme 0 5 ul 3 Prepare the reagent mixture below for the HaellI digestions and distribute 2 5 ul to the individual reaction tubes labeled for Haelll Component Volume per reaction Nuclease Free Water 1 5 ul 10x Nlalll Haelll Buffer 0 5 ul 10x Haelll enzyme 0 5 ul 4 Prepare the reagent mixture below for the Nlalll digestions and distribute 2 5 ul to the individual reaction tubes labeled for NlallI Component Volume per reaction BSA diluted to 3 33 with water 1 5 ul 10x Nlalll Haelll Buffer 0 5 ul 10x Nlalll enzyme 0 5 ul 5 Add 2 5 ul of the appropriate PCR product to the digestion reactions Vortex the reactions to mix and then briefly centrifuge the tubes 6 Incubate all the digestion reactions at 37 C for 2 hours to overnight 7 Transfer the reactions to 80 C for 20 minutes 8 Optional Add 1 ul of 60 mM EDTA to each reaction and vortex well 36 DNA Fish ID Protocol Quick Reference Protocols 3 To analyze the restriction digest patterns on a Bioanalyzer DNA 1000 Lab Chip 1 For each digest reaction pipet 1 ul of the reaction into one of the 12 sample wells on a DNA 1000 Lab chip according to the guidelines in the figure below Refer to Appendix B Agilent DNA 1000 Kit Quick St
6. a 50 ng ul dilution of the positive control salmon DNA 2 Prepare the PCR reagent mixture below Prepare enough for all your reactions plus one reaction volume excess Include a positive control reaction and a no template control reaction Component Volume 1 Reaction Volume 5 Reactions Nuclease Free Water 9 ul 45 ul 2x PCR Master Mix 12 5 ul 62 5 ul Primer Mix 2 5 ul 12 5 ul 3 Vortex the mixture then distribute 24 ul to each PCR reaction tube 4 Add 1 ul of the diluted positive control DNA to the positive control reaction tube To the test sample reactions add 1 ul of DNA sample For the no template control reaction add 1 ul of DNase free water 5 Cap the reaction tubes vortex to mix and then centrifuge briefly 6 Place the reactions in the thermal cycler and run the program below Segment Number of Cycles Temperature Duration 1 1 95 C 5 minutes 95 C 30 seconds 2 40 50 C 30 seconds 72 C 30 seconds 3 1 72 C 7 minutes 7 To analyze the PCR results on the Bioanalyzer load 1 ul of each PCR reaction on a DNA 1000 lab chip Refer to Appendix B Agilent DNA 1000 Kit Quick Start Guide on page 41 for complete instructions on preparing and running these chips on the Bioanalyzer DNA Fish ID Protocol 35 3 Quick Reference Protocols To digest PCR products with restriction enzymes 1 Label the tubes for the restriction digest reactions Each PCR product needs to be digested with 3 enzymes Ddel Haelll and
7. again in position C Retighten the screw 3 Adjust the syringe clip 44 a Release the lever of the clip and slide it down to the lowest position DNA Fish ID Protocol Appendix B Agilent DNA 1000 Kit Quick Start Guide Essential Measurement Practices e Handle and store all reagents according to the instructions on the label of the individual box e Avoid sources of dust or other contaminants Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results e Keep all reagent and reagent mixes refrigerated at 4 C when not in use e Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use e Protect dye and dye mixtures from light Remove light covers only when pipetting The dye decomposes when exposed to light and this reduces the signal intensity e Always insert the pipette tip to the bottom of the well when dispensing the liquid Placing the pipette at the edge of the well may lead to poor results e Use a new syringe and electrode cleaners with each new Kit e Use loaded chips within 5 minutes after preparation Reagents might evaporate leading to poor results e Do not touch the Agilent 2100 Bioanalyzer during analysis and never place it ona vibrating surface DNA Fish ID Protocol 5 45 5 Appendix B Agilent DNA 1000 Kit Quick Start Guide Agilent DNA 1000 Assay Protocol Edition April 2007 WARNING Handling DMSO
8. and centrifuge the tubes briefly DNA Fish ID Protocol 17 2 Procedures To run the PCR protocol 1 Place the reactions in the thermal cycler and run the PCR program shown below Table 2 PCR Cycling Protocol Segment Number of Cycles Temperature 1 1 95 C 95 C 2 40 50 C 72 C 3 1 72 C Duration 5 minutes 30 seconds 30 seconds 30 seconds 7 minutes To analyze the PCR products CAUTION At this point it is helpful to analyze the PCR products on a Bioanalyzer DNA 1000 Lab chip to ensure the product sizes are in the expected range 1 Prepare the chip Refer to Appendix B Agilent DNA 1000 Kit Quick Start Guide on page 41 for complete instructions The steps for chip priming see Loading the Gel Dye Mix on page 46 are especially critical for a successful chip run Perform these steps with care 2 Once the chip is ready start the 2100 Expert software The software opens to the Instrument context 3 Open the lid of the Agilent 2100 Bioanalyzer Check that the electrode cartridge is inserted properly and the chip selector is in position 4 Place the chip carefully into the receptacle and close the lid Forced closing of the lid may damage the electrodes and dropping the lid may cause liquid spills resulting in bad results Do not use force to close the lid and do not drop the lid onto the inserted chip 18 DNA Fish ID Protocol Procedures 5 In the Instrument context of the software
9. entry for this species to the database and include the fragment data you obtained with the positive control sample If the fragment data was imported from an XAD file be sure you clicked Reintegrate on the XAD import window DNA Fish ID Protocol 31 2 Procedures Troubleshooting Guide 32 DNA Fish ID Protocol a Agilent DNA Fish ID Ensemble Protocol 00 ee 3 Th Quick Reference Protocols To prepare the fish samples for DNA extraction 34 To extract genomic DNA 34 To set up and run the PCR reactions 35 To digest PCR products with restriction enzymes 36 To analyze the restriction digest patterns on a Bioanalyzer DNA 1000 Lab Chip 37 This chapter contains Quick Reference protocols with streamlined instructions covering the preparation of the fish samples through analysis of restriction digest patterns fhe Agilent Technologies 33 3 34 Quick Reference Protocols To prepare the fish samples for DNA extraction 1 For each test sample put a piece of fish tissue 10 mg to 1 g intoa 1 5 ml microcentrifuge tube Combine 200 ul of Proteinase K Digestion Buffer pre warmed to 65 C and 20 ul of Proteinase K for each sample to create a working solution of Proteinase K 3 Add 220 ul of the Proteinase K working solution to each fish sample Incubate the tubes at 65 C for 10 minutes Spin the tubes in a microcentrifuge for 3 5 minutes at 14 000 x g Transfer 150 ul of each supernatant into a fresh
10. fish samples for DNA extraction on page 14 To homogenize fish samples The homogenization protocol below was developed using the Omni Tissue Homogenizer but other homogenizers may also be used 1 Inacontainer suitable for homogenization combine 1 part fish tissue with 2 parts water For example mix 5 g of fish with 10 ml of water 2 Homogenize the sample until it is of a uniform consistency 3 Measure out an aliquot weighing between 40 and 200 mg This aliquot may be used in place of a fish tissue sample as the starting material in the fish identification procedures outlined in Chapter 2 DNA Fish ID Protocol Agilent DNA Fish ID Ensemble Protocol 909 Appendix B Agilent DNA 1000 Kit Quick 2 StartGuide This chapter contains a copy of the Agilent DNA 1000 Kit Quick Start Guide with instructions on preparing the DNA 1000 chips fhe Agilent Technologies M 5 Appendix B Agilent DNA 1000 Kit Quick Start Guide Assay Principles Agilent DNA kits contain chips and reagents designed for sizing and analysis of DNA fragments Each Agilent DNA chip contains an interconnected set of microchannels that is used for separation of nucleic acid fragments based on their size as they are driven through it electrophoretically Agilent DNA kits are designed for use with the Agilent 2100 Bioanalyzer only Assay Kits The Agilent DNA 1000 kit provides higher resolution of smaller fragments in comparison of our other DNA
11. sizing kits It can be used for the following applications e Analysis of PCR and RT PCR products e RFLP analyses e Heteroduplex analysis using mismatch cleavage enzymes e QC of sequencing templates The complete DNA 1000 kit guide can be found in the online help of the 2100 Expert software Storage Conditions e Keep all reagents and reagent mixes refrigerated at 4 C when not in use to avoid poor results caused by reagent decomposition e Protect dye and dye mixtures from light Remove light covers only when pipetting Dye decomposes when exposed to light Equipment Supplied with the Agilent 2100 Bioanalyzer e Chip priming station reorder number 5065 4401 e IKA vortex mixer Additional Material Required Not Supplied e Pipettes 10 ul 100 ul and 1000 ul with compatible tips e 0 5 ml microcentrifuge tubes for sample preparation e Microcentrifuge 42 DNA Fish ID Protocol Appendix B Agilent DNA 1000 Kit Quick Start Guide 5 Sample Preparation e PCR samples For accurate determination of DNA concentration the total DNA in sample must be between 0 1 50 ng ul If concentration of your particular PCR reaction is excessively high dilute to 0 1 50 ng ul in water e Restriction digests Final concentration of DNA should not exceed 50 ng ul Add EDTA and or heat inactivate the restriction enzyme according to the manufacturer instructions Restriction endonucleases in combination with non chelated metal ions may degrade i
12. tube or any oily material that may be present at the top of the tube see image below These tubes of supernatant are the samples from which genomic DNA will be extracted Supernatant To extract genomic DNA 1 Add 500 ul of Nucleic Acid Binding Buffer to each fish sample Vortex or pipet the sample repeatedly until homogenized The addition of this mixture will bring the total volume of each sample to 650 ul 2 Transfer all 650 ul of each sample to a separate DNA Binding Spin Cup that has been seated within a 2 ml receptacle tube provided and snap the cap of the tube onto the top of the spin cup 3 Spin the samples in a microcentrifuge for 1 minute at 14 000 x g to load the DNA onto the spin cup matrix 4 Remove and retain the spin cups and discard the filtrates For each sample replace the spin cup in the receptacle tube then add 500 ul of 1x High Salt Wash Buffer and cap the tube 5 Spin the samples in a microcentrifuge at 14 000 x g for 1 minute DNA Fish ID Protocol 15 2 16 Procedures Remove and retain the spin cups and discard the filtrates For each sample replace the spin cup in the receptacle tube then add 500 ul of 80 ethanol and cap the tube 7 Spin the samples in a microcentrifuge at 14 000 x g for 1 minute 8 Repeat steps 6 and 7 two more times for a total of 3 washes with 500 ul of 80 ethanol After the third wash in 80 ethanol remove and retain the spin cups and discard the filtrates R
13. Agilent DNA Fish ID Ensemble Part Number 5500 0100 Protocol Version CO June 2015 For Research Use Only Not for use in diagnostic procedures fhe Agilent Technologies Notices Agilent Technologies Inc 2015 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 5990 3062 Edition Version CO June 2015 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Rd Santa Clara CA 95051 USA Technical Support For technical product support contact Agilent at 800 894 1304 or techservices agilent com Notice to Purchaser DNA Dye Concentrate This product is provided under an agreement between Molecular Probes Inc a wholly owned subsidiary of Invitrogen Corporation and Agilent Technologies The manufacture use sale or import of this product may be subject to one or more of U S patents pending applications and corresponding international equivalents owned by Molecular Probes Inc The purchaser has the non transferable right to use the product to detect protein and or nucleic acids in microfluid ics analysis systems for one or more of the sub fields of research development quality control forensics environmental analysis b
14. ading the Markers 1 Pipette 5 ul of marker green capped tube in all 12 sample wells and ladder well Do not leave any wells empty Loading the Ladder and the Samples 1 Pipette 1 ul of DNA ladder yellow capped tube in the well marked 4 0000 2 In each of the 12 sample wells pipette 1 ul of sample used TTT 1pl ladder wells or 1 ul of de ionized water unused wells 3 Put the chip horizontally in the adapter and vortex for 1 min at the indicated setting 2400 rpm 4 Run the chip in the Agilent 2100 Bioanalyzer within 5 min Technical Support In the US Canada 1 800 227 9770 toll free lsca ibs support agilent com In Europe call your local Customer Care Center bio_solutions agilent com In Japan 0120 477 111 yan_ccr agilent com In Asia Pacific call your local Customer Care Center Bioanalyzer_ap agilent com Further Information Visit Agilent Technologies unique Lab on a Chip web site It is offering useful information support and current developments about the products and the technology http www agilent com chem labonachip DNA Fish ID Protocol 47 5 48 Appendix B Agilent DNA 1000 Kit Quick Start Guide DNA Fish ID Protocol Agilent DNA Fish ID Ensemble E Protocol e e ee 6 7 e Appendix C Bioanalyzer Electrode o gt Cleaning Procedure This chapter contains instructions on how to clean the electrodes of the Agilent 2100 Bioanalyzer Agg Agilent Tec
15. ample 10 mg 1 gram Species Identification Before You Begin Recommended Controls 11 1 Before You Begin Recommended Controls 12 DNA Fish ID Protocol Agilent DNA Fish ID Ensemble Protocol 2 Procedures To prepare the reagents 14 To prepare the fish samples for DNA extraction 14 To extract genomic DNA 15 To set up the PCR reactions 16 To run the PCR protocol 18 To analyze the PCR products 18 To digest PCR products with restriction enzymes 21 To analyze the restriction digest patterns 23 To identify the test sample species using the RFLP Decoder Software 28 Troubleshooting Guide 30 This chapter contains instructions on how to extract genomic DNA from fish samples and perform restriction fragment length polymorphism RFLP analysis using PCR amplification and restriction enzyme digestion The procedures in this chapter include detailed instructions Chapter 3 has Quick Reference protocols with streamlined instructions covering the steps in To extract genomic DNA through To analyze the restriction digest patterns fhe Agilent Technologies 13 2 Procedures To prepare the reagents High Salt Wash Buffer Prepare 1x High Salt Wash Buffer by adding 16 ml of 100 ethanol to the bottle of High Salt Wash Buffer After adding the ethanol affix the EtOH Added sticker provided to the cap Close the cap of the container tightly and store at room temperature 80 Ethanol Prepare 80 ethanol
16. art Guide on page 41 for complete instructions on preparing and running these chips on the Bioanalyzer Positive Control Sample Test Sample 1 Test Sample 2 Test Sample 3 2 D oO kes oO 0 med D lt eose 2 9 DNA Chip On Chip Electrophoresis Caliper 2 During the run the incoming raw signals are displayed in the Instrument context If necessary adjust the Peak Height Threshold setpoint 3 Import the XAD file into the RFLP Decoder application to identify the test sample fish species See To identify the test sample species using the RFLP Decoder Software on page 28 for more information or refer to the application s help system DNA Fish ID Protocol 37 3 Quick Reference Protocols 38 DNA Fish ID Protocol Agilent DNA Fish ID Ensemble Protocol ee 4 e Appendix A Homogenized Fish Samples as the Starting Material This chapter describes how to use homogenized fish samples in the fish identification protocol and instructions on fish sample homogenization Agg Agilent Technologies 39 4 40 Appendix A Homogenized Fish Samples as the Starting Material The use of homogenized fish samples In place of using a piece of intact fish tissue the fish identification protocol can be used with a homogenized sample of fish For homogenized samples use 40 to 200 mg of homogenized fish as the starting material for sample preparation see step 1 of To prepare the
17. ation can be performed in the thermal cycler If desired reactions may be left at 37 C overnight 11 Transfer the reactions to 80 C for 20 minutes This incubation can be performed in the thermal cycler 12 Optional Add 1 ul of 60 mM EDTA to each reaction and vortex well Store the reactions at 4 C until you are ready to proceed to the next step DNA Fish ID Protocol Procedures 2 To analyze the restriction digest patterns Analyze the restriction digest reactions on the Agilent 2100 Bioanalyzer to determine the fragment lengths produced during digestion 1 Pipet 1 ul of digest reaction into one of the 12 sample wells on a DNA 1000 Lab chip according to the guidelines in the figure below Refer to Appendix B Agilent DNA 1000 Kit Quick Start Guide on page 41 for complete instructions on preparing these chips Positive Control Sample Test Sample 1 Test Sample 2 Test Sample 3 N D eae O pe D lt ae Cr On Chip Electrophoresis Caliper The steps for chip priming see Loading the Gel Dye Mix on page 46 are especially critical for a successful chip run Perform these steps with care 2 Once the chip is ready start the 2100 Expert software The software opens to the Instrument context 3 Open the lid of the Agilent 2100 Bioanalyzer Check that the electrode cartridge is inserted properly and the chip selector is in position 4 Place the chip carefully into the r
18. by diluting 100 ethanol with DNase free water To prepare 80 ml of 80 ethanol enough for 50 fish samples add 16 ml of DNase free water to 64 ml of 100 ethanol To prepare the fish samples for DNA extraction 1 For each fish sample to be tested place a piece of the fish tissue raw or cooked into a single 1 5 ml microcentrifuge tube Samples ranging between 10 mg and 1 g have been used successfully but 100 400 mg samples provide the optimal yield Use the figure below as a guideline for estimating the weight of a raw fish sample based on sample size Weight mg 1000 500 200 300 100 40 20 10 Salman 7 Fish Samp Py a BB Gere Heer UTE Eat aay i by ped ka ae bea a bb aE Size cm 0 1 2 3 4 5 6 7 8 9 10 2 Pre warm the Proteinase K Digestion Buffer to 65 C for 5 minutes in an incubator or water bath 3 Prepare a working solution of Proteinase K by combining 200 ul of Proteinase K Digestion Buffer and 20 ul of Proteinase K per sample Prepare a fresh working solution of Proteinase K before each use 14 DNA Fish ID Protocol Procedures 2 4 Add 220 ul of the Proteinase K working solution to each 1 5 ml tube of fish sample Incubate the tubes at 65 C for 10 minutes in an incubator or water bath 5 Spin the tubes in a microcentrifuge for 3 5 minutes at 14 000 x g to pellet any undigested tissues 6 Transfer 150 ul of each supernatant into a fresh 1 5 ml tube Avoid transferring any undigested material from the bottom of the
19. c acids in the sample bind to the fiber matrix The immobilized nucleic acids are washed to remove contaminants and total DNA is recovered in a final volume of 100 ul The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes The PCR products are then digested with three different restriction enzymes The fragment lengths produced in these digestion reactions can be resolved on the Agilent 2100 Bioanalyzer and analyzed with the RFLP Decoder software to determine the species of fish from which the DNA sample was prepared Figure 1 summarizes the protocol for the DNA fish ID system Recommended Controls Once the DNA has been extracted from the samples of fish it is subjected to PCR amplification followed by restriction enzyme digestion We recommend including a positive control reaction and a negative control reaction alongside the test DNA samples during these steps The kit includes a genomic DNA sample isolated from Atlantic salmon that can be used as a positive control You can also use your own fish samples of known species as positive controls in the experiment DNase free water can be used in place of a DNA sample to provide a negative control Additionally when preparing the PCR reactions with the test samples we recommend setting up the reactions in duplicate DNA Fish ID Protocol DNA Fish ID Protocol Figure 1 Overview of the DNA fish ID protocol Seatood S
20. eceptacle and close the lid The chip fits only one way The electrodes in the cartridge fit into the wells of the chip CAUTION Forced closing of the lid may damage the electrodes and dropping the lid may cause liquid spills resulting in bad results Do not use force to close the lid and do not drop the lid onto the inserted chip DNA Fish ID Protocol 23 2 Procedures The 2100 Expert software screen shows that you have inserted a chip and closed the lid by displaying the chip icon at the top left of Instrument context An example screen is shown below Note that the Rest Digest check boxes in the Chip Summary panel do not need to be marked E 2100 Expert E Efx File Context View Assays Instrument Windows Help Instrument B E 3 Contexts DE34904061 DNA 1000 All Instruments Instrument Diagnostics Instrument Logbook DE34904061 Name Singulex2 ie COM Port fi Instrument 2 Y es Serial DE34904061 Assay Selection Cartridge Electrode Y a Assays gt y DE 2904879 vendor Agilent a 2 S z GC c amp lt Start Stop Run oO Start s DNA Chip Product ID G2938C verification i Taa Soer EMERE Assay File Ci NA DNA 1000 Series ILxsy LO LE Data File c laga Destination and Data Acquisition Parameters Assay Details Default C ilent 2100 bioanalyzer 2100 ex
21. ed with non fish DNA Contamination with bovine DNA for example is common in fish samples that had been mixed with milk or cheese Try using the Mixture score method to prevent mis matched fragments from impacting the combined score You may also need to repeat the DNA extraction protocol using a tissue sample with less contaminating material e g sauce breading etc The enzyme may not have completely digested the PCR product creating partially digested fragments that are longer than the expected length Examine the fragment length to see if any may be the result of incomplete enzyme digestion Review the Bioanalyzer electropherogram for the PCR reaction pre digest to see if any non specific peaks were amplified If so try excluding any fragments in the digest reaction that may be attributed to the non specific PCR product DNA Fish ID Protocol Procedures 2 Observation If the software identified multiple species as a perfect match or close match for your fish sample The software did not identify any species that are a perfect match or close match for your fish sample Suggestion If all the species identified as a potential match are highly related e g albacore tuna and yellowfin tuna the software Is likely identifying all the species together because of their similar fragment patterns You can try lowering the Match Tolerance setting until only one species is a close or perfect match but also consider what you kno
22. eplace the spin cups in their receptacle tubes and spin in a microcentrifuge for 2 minutes at 14 000 x g to dry the fiber matrix 10 Transfer the spin cups to fresh 1 5 m1 collection tubes Add 100 ul of Elution Buffer to each spin cup directly on the fiber matrix inside the cup Snap the caps of the collection tubes onto the spin cups and incubate at room temperature for 1 minute 11 Spin the samples in a microcentrifuge at maximum speed for 1 minute 12 The purified DNA is in the Elution Buffer in the microcentrifuge tube Discard the spin cups and cap the tubes The DNA may be stored at 4 C for up to one month For long term storage store the DNA at 20 C or 80 C 13 If desired you may measure the concentration of the DNA samples in a spectrophotometer The genomic DNA extraction protocol typically yields samples with a concentration ranging from 5 ng ul to 500 ng ul The PCR RFLP protocol works wells with DNA samples ranging anywhere from 0 05 ng ul to 2000 ng ul To set up the PCR reactions 1 Prepare a 50 ng ul dilution of the positive control salmon DNA by combining 8 ul of the DNA stock with 32 ul of sterile DNase free water Vortex briefly to mix Keep the diluted sample on ice while setting up reactions The diluted sample may be stored at 4 C for future use Prepare the reactions on ice by combining the components in Table 1 in order Prepare a single reagent mixture for all PCR reactions that will be ru
23. es To identify the test sample species using the RFLP Decoder Software The Agilent software application RFLP Decoder may be used to identify the fish species for the test DNA samples based on the fragment lengths produced in the digestion reactions These instructions provide the basics on use of the RFLP Decoder software Refer to the software s help system for detailed information on operating the software and interpreting the display 1 Launch the RFLP Decoder program 2 Click File gt Open gt XAD File The Open dialog box will open 3 Select the XAD file for the DNA chip that included the restriction digest reactions Click Open The data from the XAD file are loaded into the Bioanalyzer import table on the right side of the screen 4 In the field labeled Min Peak height as of lower the recommended value is 10 0 If needed you may use a lower value to identify small peaks that were missed or a higher value to discard peaks resulting from non specific noise in the electropherogram Click Reintegrate before proceeding or mark the check box for Automatic Reintegration Integrator Min Peak hei as ie print 10 0 Gy X Reintegrate L Automatic Reintegration 5 In the Bioanalyzer import table select three digestion reactions corresponding to one DNA sample and specify the appropriate restriction enzyme for each well using the drop down lists under the Enzyme column In the figure below the Enzyme column has been
24. filled out for the Atlantic salmon sample Enzyme FI Atlantic salmon D Ddel x 121 Atlantic salmon H Haei x 105 Mlantic salmon N Nalli 7 465 SS a Se 8 g S a 28 DNA Fish ID Protocol DNA Fish ID Protocol Procedures 6 Near the bottom right corner of the screen click Calculate The fragment length data obtained from the Bioanalyzer import table will populate the fields in the fragment data panes tabs on the left side of the screen In the Score drop down list near the top left corner of the screen select the appropriate algorithm for analysis of the data If the fish sample being analyzed consists of a single fish species select Dice Nei Li in the Score drop down list If the samples may consist of a mixture of species select Mixture The table labeled Combined score lists the best species matches based on the results of all three digestion reactions In the Lower Cutoff and Match Tolerance fields in the analysis parameters at the top of the screen you may adjust the settings to improve the score of the best species match e The Lower Cutoff value is used to discard any fragments shorter than the length designated in the field The default is 30 bp e The Match Tolerance value determines how close in length a fragment must be to the predicted fragment to be considered a match The default is 10 Repeat steps 5 through 8 for the remaining DNA samples that were included on this same chip
25. hapter and have the necessary equipment and reagents listed before you start an experiment Procedures This chapter contains instructions on how to extract genomic DNA from fish samples and perform restriction fragment length polymorphism RFLP analysis using PCR amplification and restriction enzyme digestion Quick Reference Protocols This chapter contains Quick Reference protocols with streamlined instructions covering the preparation of the fish samples through analysis of restriction digest patterns Appendix A Homogenized Fish Samples as the Starting Material This chapter describes how to use homogenized fish samples in the fish identification protocol and instructions on fish sample homogenization Appendix B Agilent DNA 1000 Kit Quick Start Guide This chapter contains a copy of the Agilent DNA 1000 Kit Quick Start Guide with instructions on preparing the DNA 1000 chips Appendix C Bioanalyzer Electrode Cleaning Procedure This chapter contains instructions on how to clean the electrodes of the Agilent 2100 Bioanalyzer References DNA Fish ID Protocol Contents 1 2 DNA Fish ID Protocol Before You Begin 7 Materials Provided with the Agilent DNA Fish ID Ensemble Storage Conditions 9 Required Equipment Supplies and Reagents 9 Overview of the Agilent DNA Fish ID Solution 10 Recommended Controls 10 Procedures 13 To prepare the reagents 14 To prepare the fish samples for DNA extraction 14 To ext
26. hnologies 49 6 Appendix C Bioanalyzer Electrode Cleaning Procedure To Clean Electrodes after a DNA 1000 Chip Run Use a new electrode cleaner with each new DNA 1000 kit CAUTION Liquid spill might cause leak currents between the electrodes Never fill too much water in the electrode cleaner 1 Slowly fill one of the wells of the electrode cleaner with 350 ul deionized analysis grade water 2 Open the lid and place the electrode cleaner in the Agilent 2100 Bioanalyzer 3 Close the lid and leave it closed for about 10 seconds 4 Open the lid and remove the electrode cleaner 5 Wait another 10 seconds to allow the water on the electrodes to evaporate before closing the lid After 5 assays empty and refill the electrode cleaner After 25 assays replace the used electrode cleaner with a new one When switching between different assays a more thorough cleaning may be required Refer to the Maintenance and Troubleshooting chapter in the 2100 Expert help system 50 DNA Fish ID Protocol Agilent DNA Fish ID Ensemble Protocol References and Publications 1 Dooley John J Sage Helen D Brown Helen M Garrett Stephen D Improved fish species identification by use of lab on a chip technology Food Control 16 7 601 607 2004 Dooley John J Sage Helen D Clarke Marie Anne L Brown Helen M Garrett Stephen D Fish Species Identification Using PCR RFLP Analysis and Lab on a Chip Capillary Electrophores
27. iodefense food safety testing veterinary diagnostics or human diagnostics according to use indicated on the product label or accompanying product litera ture For information on obtaining a license con tact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 9132 Tel 541 465 8300 Fax 541 335 0354 Warranty The material contained in this document is provided as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc ument that conflict with these terms the warranty terms in the separate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restric
28. is Application to Detect White Fish Species in Food Products and an Interlaboratory Study J Agric Food Chem 53 3348 3357 2005 Dooley J Garrett S Determination of PCR RFLP Profiles for Fish Species Using the Agilent 2100 Bioanalyzer Agilent Technologies Application Note 2005 Dooley J J Clarke M L Sage H D Brown H M Garrett S D Application of a chip based capillary electrophoresis system to enable simple PCR RFLP identification of fish species FSA Final Report Q01069 2004 fhe Agilent Technologies 51 www agilent com In This Book This document describes how to use the Agilent DNA Fish ID Ensemble to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis Agilent Technologies Inc 2015 Version C0 June 2015 5990 3062 fhe Agilent Technologies
29. lent PCR RFLP Fish Species ID Kit Part Number 5500 0001 100 reactions Positive Control Salmon DNA 250 ng ul 10 ul 2x PCR Master Mix 1 25 ml Nuclease Free Water 2 ml 10x Fish Species ID Primer Mix 250 ul 10x Nlalll Restriction Enzyme 50 ul 10x Ddel Restriction Enzyme 50 ul 10x Haelll Restriction Enzyme 50 ul 10x Nlalll Haelll Buffer 100 ul 10x Ddel Buffer 50 ul BSA 100x solution 100 ul 60 mM EDTA 300 ul Agilent DNA 1000 Kit Part Number 5067 1504 25 chips DNA Chips 25 each Electrode Cleaner 1 each Syringe 1 each Spin Filters 3 each DNA 1000 Ladder yellow capped tube 35 ul DNA 1000 Markers 15 1500 bp green capped tube 2x1 2ml DNA Dye Concentrate blue capped tube 90 ul DNA Gel Matrix red capped tube 3 x 500 ul Nia Ill is licensed under U S Patent No 5 278 060 Haelll is licensed under U S Patent No 5 179 015 See Notice to Purchaser DNA Dye Concentrate on page 2 for licensing information 8 DNA Fish ID Protocol Storage Before You Begin Conditions Store the proteinase K and proteinase K digestion buffer at 4 C Store the remaining DNA isolation kit components at room temperature Store the components of the PCR RFLP reagent kit at 20 C upon receipt Once thawed store the 10x Primer Mix at 4 C Continue storing the remaining PCR RFLP components at 20 C Store all components of the DNA 1000 Kit at 4 C Protect dye and dye mixtures from light remove ligh
30. max 16 characters Run sample 1 to 12 7 Click the Start button to start the chip run 24 DNA Fish ID Protocol CAUTION Procedures 2 After each run immediately remove the used chip from the Bioanalyzer and dispose of it according to good laboratory practice Then perform the electrode cleaning procedure described in Appendix C Bioanalyzer Electrode Cleaning Procedure on page 49 DNA Fish ID Protocol 8 When the run is complete peaks are identified for all samples using the settings of the peak find algorithm Review the electropherogram in the Data context to determine if any genuine peaks have been missed or if any non specific peaks have been erroneously identified e If you believe the algorithm has failed to detect a genuine peak you may lower the Height Threshold setpoint for that well to a value that allows the algorithm to identify the peak To lower the threshold click View gt Setpoints and with the Local tab selected enter a new value into the Height Threshold FU field Local Global Normal Collapse Integration Start Time s 30 Integration End Time s 128 95 Slope Threshold 0 5 Area Threshold 0 1 Height Threshold FU 20 Width Threshold s 0 5 e Occasionally non specific peaks appear on the electropherogram as dramatic spikes see below for an example If the electropherogram includes a peak such as this exclude the peak from analysis To exclude a peak turn on Manual I
31. n simultaneously by scaling up the volumes listed in the table DNA Fish ID Protocol Procedures 2 In addition to the test DNA samples include a positive control reaction and a no template control reaction Preparing duplicate PCR reactions for each test DNA sample is recommended Prepare enough reagent mixture for all your reactions plus one reaction volume excess For example if you have 5 test DNA samples prepare enough reagent mixture for either 8 reactions 5 test reactions 1 positive control 1 no template control and 1 excess or 13 reactions if you are including duplicates of the test reactions 10 duplicate test reactions 1 positive control 1 no template control and I excess Table 1 PCR Reagent Mixture Volume Volume Component 1 Reaction 5 Reactions Nuclease Free Water 9 ul 45 ul 2x PCR Master Mix 12 5 ul 62 5 ul Primer Mix 2 5 ul 12 5 ul Total volume 24 ul 120 ul 3 Vortex the reagent mixture well then distribute 24 ul to each individual thin walled PCR reaction tube 4 Add 1 ul of the diluted positive control DNA to the positive control reaction tube To the test sample tubes add 1 ul of test DNA sample For the no template control reaction add 1 ul of DNase free water in place of the DNA To avoid cross contamination use a fresh pipet tip for each DNA sample After adding the sample mix the reaction by quickly pipetting the contents of the tube up and down 5 Cap the reaction tubes vortex the tubes to mix
32. ntegration select the peak on the electropherogram then right click and select Exclude Peak A K s 100 Non specific peak S Qy 4 e 15 SO 100 150 200 300 400 500 700 1500 bp 25 2 Procedures To analyze the restriction digest patterns 9 Go to the Assay context and select the Chip Summary tab In the Sample Name field enter a sample name for all 12 wells on the chip as shown in the figure below Positive Control Ddel o 2 Positive Control Hael Positive Control Miall OOOO 4 DNA Sample Ddel OO o 5 DMA Samplet Hael OOOO DMA Sampled Mall OOOO 7 DNA Sample Ddel Oo o 8 DMA Sample Hael O OOOO DMA Sample Mall OOOO 10 DNA Sample Ddel oOo 11 DMA Samples Hael OOOO DMA Samples Miall O An image of a Bioanalyzer gel with restriction digest samples from four different fish species is shown in Figure 3 The expected results for the positive DNA sample are summarized in Table 7 26 DNA Fish ID Protocol Procedures 2 Figure 3 Bioanalyzer gel image of restriction digest reaction samples Each gel lane is labeled with the restriction enzyme used in that reaction Trout Tuna Rock sole Pacific cod Ddel Haelll Nialll Ddel Haelll Nialll Ddel Haelll Nlalll Ddel Haelll Nlalll Table 7 Expected DNA Fragment Sizes in the Salmon Positive Control Restriction Enzyme Expected Product Size bp Ddel 117 332 340 Haelll 40 105 333 Nialll 459 DNA Fish ID Protocol 27 2 Procedur
33. nternal DNA markers used in assay kit Physical Specifications Type Specifications Analysis run time 35 minutes Number of samples 12 samples chip Sample volume 1 ul Kit stability 4 months Storage temperature see individual box Analytical Specifications Type Agilent DNA 1000 Assay Sizing range 25 1000 bp Typical sizing resolution 5 bp 25 100 bp 5 100 500 bp 10 500 1000 bp Sizing accuracy 10 for ladder as sample Sizing reproducibility 5 CV for ladder as sample Quantitation accuracy 20 CV for ladder as sample Quant reproducibility 25 500 bp 15 CV 500 1000 bp 5 CV for ladder as sample Quantitative range 0 1 50 ng ul Maximum salt 250 mM for KCI or NaCl 15 mM for MgCl Some fragments below 70 bp may deviate from the above specifications DNA Fish ID Protocol 43 5 Appendix B Agilent DNA 1000 Kit Quick Start Guide Setting up the Chip Priming Station 1 2 Replace the syringe a Unscrew the old syringe from the lid of the chip priming station b Release the old syringe from the clip Discard the old syringe c Remove the plastic cap of the new syringe and insert it into the clip d Slide it into the hole of the luer lock adapter and screw it tightly to the priming station Adjust the base plate a Open the chip priming station by pulling the latch b Using a screwdriver open the screw at the underside of the base plate c Life the base plate and insert it
34. ontrol salmon DNA 490 bp 10 Test samples 490 bp 10 No template control No product the presence of product in the no template control is an indication of contamination or primer dimer DNA Fish ID Protocol Procedures To digest PCR products with restriction enzymes DNA Fish ID Protocol Once the PCR is complete the PCR reactions are treated with restriction enzymes for restriction fragment length polymorphism RFLP analysis 1 Label the 0 5 ml tubes or 0 2 ml strip tubes that are to be used for the restriction digest reactions Each PCR reaction will be digested with three different restriction enzymes Ddel HaellI and Nlalll Therefore for each PCR reaction label three separate tubes with the name of the PCR sample and the name of the restriction enzyme 2 Prepare the reagent mixture for the Ddel digestions by combining the components in Table 4 in order Prepare a single reagent mixture for all Ddel digestion reactions plus at least one reaction volume excess using multiples of each component Table4 Ddel Digestion Reagent Mixture Component Volume Nuclease Free Water 1 5 ul 10x Ddel Buffer 0 5 ul 10x Ddel enzyme 0 5 ul 3 Vortex the reagent mixture well then distribute 2 5 ul to the individual reaction tubes that were labeled for Ddel 4 Prepare the reagent mixture for the HaellI digestions by combining the components in Table 5 in order Prepare a single reagent mixture for all Haelll digestion reaction
35. pert D ata 201 0 03 18 Assay Class DNA 1000 a Custom C lent2100 bioanalyzer2100 expert D ata2010 03 18 aalr pe 02 2009 q File Prefix 2100 expert max 16 characters 9 48 52 AM Run sample j itof 12 aa Comments DNA Analysis 25 1000 bp System x v EL gt m Chip Summary Start Run Checklist Sample Name Sample Comment Rest Digest Observal of Is the instrument ready gt Sample 1 O of Is a chip detected 2 Sample 2 m f Is selected instrument valid for this asse 3 Sample 3 O of Does the cartridge and the selected ass 4 Sampe4 H of Are all required licenses applied 5 Sample 5 O of Is the instrument type supported 6 Sample 6 Oo f Is the required firmware version definec 7 Sample 7 E 8 Sample 8 O Ta 9 Sample 9 O em Loan ea i set gt EEE Le Connected to DE34904061 at COM 1 Auto Export Auto Print Auto Run 5 In the Instrument context of the software select the appropriate assay from the Assay menu Assay gt Electrophoresis gt dsDNA gt DNA 1000 Series II xsy 6 Accept the current File Prefix or modify it Data will be saved automatically to a file with a name using the prefix you entered At this time you can also customize the file storage location Destination and Data Acquisition Parameters f Default Coy lenty2 100 bioanalpzer s27 00 espert O ata 201 0 03 16 E Custom Cy lent 2100 broanalyzer 21 00 espert D ata201 0 03 18 File Prefix 2100 expert i
36. ract genomic DNA 15 To setup the PCR reactions 16 To run the PCR protocol 18 To analyze the PCR products 18 To digest PCR products with restriction enzymes 21 To analyze the restriction digest patterns 23 8 To identify the test sample species using the RFLP Decoder Software 28 Troubleshooting Guide 30 Quick Reference Protocols 33 Appendix B Agilent DNA 1000 Kit Quick Start Guide 41 Appendix C Bioanalyzer Electrode Cleaning Procedure 49 References 51 Appendix A Homogenized Fish Samples as the Starting Material 39 Contents 6 DNA Fish ID Protocol a Agilent DNA Fish ID Ensemble Protocol c 00 oes 1 Before You Begin r Materials Provided with the Agilent DNA Fish ID Ensemble 8 Storage Conditions 9 Required Equipment Supplies and Reagents 9 Overview of the Agilent DNA Fish ID Solution 10 Recommended Controls 10 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment fhe Agilent Technologies 1 Before You Begin Materials Provided with the Agilent DNA Fish ID Ensemble Agilent DNA Isolation Kit Part Number 5500 0051 50 preparations Nucleic Acid Binding Buffer 25 ml High Salt Wash Buffer 24 ml Elution Buffer 10 mM Tris HCl pH 7 5 12 ml DNA Binding Spin Cups and 2 ml Receptacle Tubes 50 each 1 5 ml Collection Tubes 50 each Proteinase K 2 x 0 5 ml Proteinase K Digestion Buffer 2 x 5ml Agi
37. s plus at least one reaction volume excess using multiples of each component Table 5 Haelll Digestion Reagent Mixture Component Volume Nuclease Free Water 1 5 ul 10x Nlalll Haelll Buffer 0 5 ul 10x Haelll enzyme 0 5 ul 21 2 2 22 Procedures 5 Vortex the reagent mixture well then distribute 2 5 ul to the individual reaction tubes that were labeled for Haelll Prepare the reagent mixture for the Nlalll digestions by combining the components in Table 6 in order Prepare a single reagent mixture for all NlallII digestion reactions plus at least one reaction volume excess using multiples of each component Table 6 Nialll Digestion Reagent Mixture Component Volume BSA diluted to 3 33x 1 5 ul 10x Nlalll Haelll Buffer 0 5 ul 10x Nlalll enzyme 0 5 ul Prepare the 3 33x BSA dilution by combining 1 ul of the provided 100x BSA stock with 29 ul of sterile nuclease free water Diluted BSA may be stored at 20 C for future use Vortex the reagent mixture well then distribute 2 5 ul to the individual reaction tubes that were labeled for NlallII For each digestion reaction add 2 5 ul of the appropriate PCR product to the labeled tubes All of the test PCR reactions as well as the positive control reaction need to be digested with all three restriction enzymes 9 Vortex the digestion reactions and then briefly centrifuge the tubes 10 Incubate all the digestion reactions at 37 C for 2 hours This incub
38. t covers only when pipetting Required Equipment Supplies and Reagents DNA Fish ID Protocol Additional Equipment Supplies and Reagents Required 100 ethanol 200 proof USP grade or equivalent Sterile nuclease free water Pipettes 10 ul 100 ul and 1000 ul with compatible tips Incubator or water bath set to 65 C Sterile glass bottle or polypropylene tube e g 14 ml BD Falcon polypropylene round bottom tubes or 50 ml BD Falcon polypropylene conical tubes Vortex mixer Thermal cycler Thin walled PCR tubes Microcentrifuge Microcentrifuge tubes Agilent 2100 Bioanalyzer with PC chip priming station and IKA vortex mixer Agilent RFLP Decoder Software 1 1 10 Before You Begin Overview of the Agilent DNA Fish ID Solution The Agilent DNA Fish ID solution is a simple fast and accurate DNA based method to identify the fish species present in a seafood sample The method consists of purification of genomic DNA from fish samples and amplification of this DNA in a PCR reaction using primers that recognize a specific region of the fish genome The PCR products are then used in restriction fragment length polymorphism RFLP analysis to identify the sample Protocol Summary The samples are first treated with proteinase K to release the nucleic acids into solution DNA is then isolated by suspending the sample in binding buffer and loading onto a micro spin cup containing a silica based fiber matrix The nuclei
39. ted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met DNA Fish ID Protocol In this Guide DNA Fish ID Protocol This document describes how to use the Agilent DNA Fish ID Ensemble to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis Before You Begin Make sure you read and understand the information in this c
40. w about the sample such as where the fish was caught to determine which of the potential matches Is the correct one If several of the fragments included in analysis are potentially non specific increase the minimum peak height threshold to exclude these fragments Review the Bioanalyzer electropherograms for the digest reactions Could any of the peaks being used for fragment analysis be background noise If so try excluding those peaks from analysis Does the electropherogram show any peaks that are below the minimum peak height threshold but may actually be real products of the digest Try lowering the threshold to include these smaller peaks Review the Bioanalyzer electropherogram for the PCR reaction pre digest to see if any non specific peaks were amplified If so try excluding any fragments in the digest reaction that may be attributed to the non specific PCR product Exclude any fragments that may be primer dimers These fragments will generally be around 68 bp long Is it possible that your fish sample contains more than one species or is contaminated with non fish DNA e g from milk or cheese present in the sample If so be sure the Mixture score method is selected The sample could be from a fish species that is not in the reference database Iry using the theoretical database to see if a match is found If you have an idea of what the species may be run a positive control sample with a fish of that species Then add an
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