SP003-OmniAmp-Supplemental Protocol
Contents
1. C No increase in assay fluorescence above background is detected Functional Assays OmniAmp Isothermal Amplification system is tested for performance by isothermal amplification of regions of the MS2 bacteriophage RNA genome and the E coli DNA genome The resulting amplification products are visualized on ethidium bromide stained agarose gels 12 SP003 Rev A Appendix D Notice of Limited Label License Copyright Patents Warranties Disclaimers and Trademarks Copyright 2013 by Lucigen Corp All rights reserved Lucigen and OmniAmp are registered trademarks of Lucigen Corp Lucigen s products are sold for research use only and are not to be used in humans or for medical diagnostics Lucigen s liability with respect to any OmniAmp product is limited to the replacement of the product No other warranties of any kind expressed or implied including without limitation any implied fitness for any particular use are provided by Lucigen Lucigen is not liable for any direct indirect incidental or consequential damages arising out of or in connection with the use or inability to use any of its products Some applications in which Lucigen enzymes can be used may be covered by other patents issued and applicable in the United States and certain other countries Because purchase of this product does not include a license to perform any patented application users of this product may be required to obtain a patent license depending upon the2. amplification guidelines please go to page 9 LAMP Reaction Optimization Lucigen recommends optimizing reaction conditions in two separate steps In the first step determine the optimal Magnesium and Betaine concentrations In the second step use that buffer formulation identified in step 1 over a range of temperatures to find the condition with the best overall performance Step 1 Magnesium and Betaine Concentration Magnesium and Betaine concentrations can be easily optimized using an array of reactions run in parallel For best results use a 96 well plate in a calibrated thermocycler For buffer optimization work perform all reactions at 68 C Note Polymerase Buffer C already contains MgSO at 2 mM which is not sufficient for most reactions Therefore most reactions will require supplemental magnesium sulfate Use of buffers other than Buffer C provided in this kit is not recommended Note To allow more precise adjustments for individual reactions the stock 5 M Betaine solution can be diluted to 1 M using nuclease free water Using these suggested increments in a matrix of conditions it should be possible to quickly find an approximately optimal reaction formulation More precise optimization can be done in subsequent steps if needed SP003 Rev A Step 2 Temperature Optimization After the optimum buffer composition has been established determine the best temperature for your targets by performing the reaction at a ran
3. 1 2613 b 4titude 4ti 0790 0 2 mL Single Tube flat cap SP003 Rev A 5 RNase free environment and procedures should be used to avoid contamination 6 Add target in an area separated from the area where the reaction mix is prepared 7 Thaw reagents and set up reactions on ice 8 Reaction setup should be done using good laboratory techniques that minimize cross contamination Note First time users are strongly encouraged to perform the control reaction as described below Table 1 in order to ensure successful results with LAMP and the OmniAmp system Control Reaction Table 1 aures SS T T Poses OP To o Positive control can be used at dilutions from 1 5 to 1 500 with reliable results Workflow In order to minimize cross contamination steps 6 and 7 should be done in an area separate from area where you are preparing reaction mix 1 Thaw all kit components and hold on ice 2 All components should be mixed well before use Vortex all tubes for 10 seconds then centrifuge briefly to collect 3 Prepare the reaction mix as shown in Table 1 in the order listed Add all the components except the template Positive Control During this step the reaction mix tube should always be held on the ice to prevent background activity of enzyme 4 Once all reagents have been added mix the reaction completely This step is required to ensure uniform distribution of all reaction components 5 Dispense 24 uL of reaction mix in
4. 2 QIAGEN Lake ConstanceProduct Information e SpeedxXtract Nucleic Acid Kit https www qiagen com gb about us contact oem services ese instruments speedxtract nucleic acid kit e ESEQuant TS2 https Awww giagen com gb about us contact oem services ese instruments esequant ts2 For more information on Qiagen solutions contact QIAGEN Lake Constance GmbH Jacques Schiesser Str 3 78333 Stockach Germany Tel 49 7771 91660 Fax 49 7771 9166218 E mail info qlc giagen com 10 SP003 Rev A Appendices Appendix A Examples of Target Specific and Nonspecific LAMP Amplification Correct target specific amplification Figure 1 Lane 1 100 bp Marker Lane 2 Negative LAMP reaction Lane 3 and Lane 4 Positive LAMP reaction products from an RNA Control template A distinct banding pattern is seen among the smear Figure 2 Lane 1 100 bp ladder Lane 2 and Lane 3 Background Amplification in a LAMP reaction Non specific or Background amplification appears as a single continuum of fragments with no visible or indistinct bands A prominent primer dimer band is also characteristic of non specific amplification Lane 4 Negative LAMP reaction Appendix B LAMP Resources 11 SP003 Rev A Eiken PrimerExplorer Software The Eiken PrimerExplorer software is an online software application that will assist users in designing a LAMP primer set The software can be accessed at the URL listed below For convenience the user man
5. 2 800 uM dNTPs 25 mM each ee p08 100 mM MgSO 2 12 mM 1 0 2 0 8 mM 2 uL 5 M Betaine 0 1 0 5M 0 5 2 5 0 15 M 0 75 uL Dye 20X Target Specific Primer Mix 10X Variable e ae OmniAmp DNA Polymerase 50X 1X 2X Template RNA or DNA 0 01ng 100 ng Buffer C is prepared with low magnesium 2 mM final to allow aT However most LAMP systems will require a final MgSO4 concentration of 6 mM or greater o 4 pD Se SP003 Rev A It is strongly recommended to use dNTPs that have not undergone multiple freeze thaw cycles LAMP systems are more sensitive to dNTP quality than typical PCR systems so fresh dNTP s are recommended for applications where sensitivity and reproducibility are important Please see Appendix B LAMP Resources for information on LAMP Primer design Note If a dye is added for amplification quantitation use between 0 2 uL and 1 0 uL per reaction or other per the manufacturer s recommendations Excessive amounts of dye will interfere with or inhibit the reaction Isothermal Amplification Conditions The following general program is recommended Step Temperature Time __ 1 Amplification 68 C 72 C 20 35 Minutes 2 Hold 4 C eo The amplification threshold is usually reached in 8 20 minutes depending on template concentration However long reactions can lead to undesired background Please see the optimization notes below on reaction time For additional
6. 881901 1 1 mL LAMP Kit RNA Control LAMP Primer Mix 10x 8129441 20 HL Nuclease free Water F98698 1 1 mL Positive Control F823398 1 20 uL 500 OmniAmp DNA Reactions ante Polymerase 50X peel anee Sapph SP003 Rev A 10X DNA Polymerase F881958 1 5x1 mL Buffer C Magnesium Sulfate 100 mM F98695 1 5x1mL Betaine 5 M F881901 1 5x1mL RNA Control LAMP Primer Mix 10x 812944 1 5 x 20 uL Nuclease free Water F98698 1 5x1 mL Positive Control F823398 1 5 x 20 uL Components and Storage Store all kits and components at 20 C 15 C max 25 C min 1 Material to be Supplied by the User dNTP mix 25 mM each 20X EvaGreen Dye Biotium Target specific 10X LAMP Primer mix Common formulation 16 uM FIP and BIP primers 8 uM Loop F and Loop B primers and 2 uM F3 and B3 primers Target RNA or DNA Reaction Setup Before you start 1 Always wear gloves while handling the components 2 Verify sufficient volume of kit components required for planned reactions prior to setup 3 Set up and protocol described below is using real time thermocycler If using a heat block replace amount of dye with Nuclease free H20 4 Make sure that the ESEQuant TS2 is set to desired temperature and acticate channel 470 520 We recommend using the following off the shelf tubes with the ESEQuant TS2 a VWR PCR strip with attached Optically Clear Cap volume 0 2 mL color natural Cat No 21
7. Nie Lucigen Simplifying Genomics OmniAmp RNA amp DNA LAMP Kit Supplemental Protocol This protocol is recommended for use with the ESEQquant TS2 real time isothermal amplification device QIAGEN Lake Constance GmbH FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE IMPORTANT 20 C Storage Required Immediately Upon Receipt SP003 Rev A Table of Contents Table Of Content i iii annann aana nananana aaae aaa aa naaa aa eaaa aaa aa aaa aaa a aa a Aaaa aE 2 Technical SUPPO t sec sccsccssecesscesssesscrdesciesesusocedetesscutscesessdesseuieiavssessdesscesedcessreniscsesiedsdersceiesceeseduestentessiens 2 Product DESCHIPTON ii cicscivessccscasecaceneraresectandteccsecevaued cancuadenecavacasadecieuencterarereaeueacaecaceuaraiedieudcatereuetevauince 3 Product Designations and Kit Components ccsecceeeeeeeeeeeeeeeseeeeeneeseeeneeeseneeseeeneneeseeeseeneeeeseeeeees 3 COMPONENTS and Storage seisieiaceaccssccnssastsastassncssnaiensansndsanccaucassncsncesnasansacranteancencnedancanedssneacadeaaasaaceaaats 4 Material to be Supplied by the USEP sciiviiivesccccenicinc nce sninanesniecnnennsanssnnewnnewenecaceennuan vane snuewnunuemesuienunaniy 4 Before YOu Stalt cccccseecssseeseeeeeseneesseneenseeeeseneesseeeeseeeeeseneeaseeeeaseeeeseneeaseeeeaseeeeseneeaseeeeeseeeeneneeaseeeenseeeeees 4 Reaction SQUID isiccecatecedcedidacdudecucatacesitecasecsdecideseiadisadesusscasucaducaducavadeseducataculedadaceiudavadsselacnl cndcsduaenn
8. a PCR tube or 96 well PCR plate for each reaction SP003 Rev A 6 On ice prepare 1 5 dilution of Positive Control by combining 1 uL of it with 4 uL of Nuclease free Water 7 Add 1 uL of the 1 5 diluted Positive Control to each well tube 8 Cap tubes or seal plate wells Centrifuge briefly to collect prior to incubation 9 Place tubes into into the ESEQuant TS2 instrument and start run at 70 C for 30 minutes using channel 470 520 for detection 10 Incubate at 70 C for 30 minutes 11 If required run samples on a 2 agarose gel Note Reactions may be kept at 20 C for longer term storage Target Specific Optimization OmniAmp polymerase is provided with Polymerase Buffer C which is designed to support LAMP and other isothermal amplification processes Buffer C contains all components required for amplification including Magnesium Sulfate MgSO at 2 mM final concentration However certain targets and amplification systems will require optimization using the included MgS0O4 and Betaine supplements For more information on optimization see LAMP Reaction Optimization section below For most targets optimization of Magnesium and Betaine concentrations will result in shorter time to result and reduced background amplification See Appendix A Examples of Target Specific and Nonspecific LAMP Amplification Experimental Reaction Final Concentration Recommended Nuclease free H2O 10X DNA Polymerase Buffer C 282
9. an hands and skin Avoid introducing RNases rather than trying to remove them Some basic precautions must be taken to work successfully with RNA e Always wear gloves to prevent introducing RNase contamination from human hands e Change gloves frequently especially after touching skin door knobs and common surfaces e Use a set of pipettors dedicated solely for RNA work e Use RNase free plasticware and reagents e Designate an RNAse free area of the lab Cold Reaction Set Up The OmniAmp polymerase has residual activity above 4 C that can cause non specific background amplification at temperatures below specific reaction temperature of 66 to 72 C e All reactions using OmniAmp Polymerase should be set up on ice and maintained at 4 C prior to amplification e Primers should be added just prior to target addition and incubation Template Preparation Most routine methods of template purification are sufficient e g phenol chloroform or guanidine silica based methods However trace amounts of purification agents phenol EDTA Proteinase K ethanol etc may inhibit amplification It is preferred that the nucleic template be dissolved in water or EDTA free buffer rather than TE following purification If TE is required formulation with 0 1 mM EDTA will give best results Reaction Overlay A thermal cycler with a heated lid is ideal to prevent evaporation of the reaction mix If no such lid is available the reaction mixture can b
10. conditions of the Limited Label License The purchase price of this product includes limited nontransferable rights to use only the purchased amount of the product and only as described in the Kit Instruction Manual This limited license specifically excludes manufacture of OmniAmp DNA Polymerase or any derivatives thereof The buyer cannot modify this product for any purpose without express written consent of Lucigen Corp Lucigen Corporation reserves all other rights in particular the purchaser of this product may not transfer or otherwise sell this product or its components or derivatives to a third party and no rights are conveyed to the purchaser to use the product or its components or derivatives for commercial purposes The buyer may transfer information or materials made through the employment of this product or its components to a scientific collaborator provided that such transfer is not for commercial purposes and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for commercial purposes Commercial purposes includes any activity for which a party receives consideration and may include but is not limited to 1 use of the product or its components or derivatives in manufacturing 2 use of the product or its components or derivatives for diagnostic purposes 3 use of this product or materials made there
11. e overlaid with one half reaction volume of PCR grade mineral oil This may slow the reaction dNTP s For best results or when sensitivity or reproducibility are critical use a stock of dNTP s that have not undergone multiple freeze thaws LAMP systems can be more sensitive than PCR to the quality of dNTP s Dye for Quantitation If you intend to add a reagent for quantitation of the reaction or measurement of its progress be aware that excessive dye may inhibit the reaction Conditions will vary and will require optimization but dye should be used at or below common working concentrations References Notomi T Okayama H Masubuchi H Yonekawa T Watanabe K Amino N Hase T Loop mediated isothermal amplification of DNA Nucleic Acids Res 2000 28 12 E63 SP003 Rev A Nagamine K Hase T Notomi T Accelerated reaction by loop mediated isothermal amplification using loop primers Mol Cell Probes 2002 16 3 223 9 Mori Y Nagamine K Tomita N Notomi T Detection of loop mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation Biochem Biophys Res Commun 2001 289 1 150 4 Mori Y Hirano T Notomi T Sequence specific visual detection of LAMP reactions by addition of cationic polymers BMC Biotechnol 2006 6 3 Tomita N Mori Y Kanda H Notomi T Loop mediated isothermal amplification LAMP of gene sequences and simple visual detection of products Nat Protoc 2008 3 5 877 8
12. eninans 4 Farget SpeciiC OpumMiZAllOn sissie te ceceteie tata iasi reden eeaeee tetateteteatetatetatateaatecntcteietenuaetatass 6 Experimental R AClONs 2 8 t2nea cnn anda aad a Ae a a a a ea 6 Gol g 170 itm 12 0 16 reer reeeeereeer ry er rest n a a ot rr er cvemer perce Tren reenter lt et rete arererruerrescerereer reer er 5 Isothermal Amplification Conditions c cccccccceesseceenceeeneeeseneeceneeseaaeesensecsneeesaeessaeeesenseeseaeeesnsseneneas 7 LAMP Reaction Op timiZatiO i acmacicniinnssccnncsancternetnenscnausnnietusninietmusintetenntunetonntaisiernieiatoanentetaiwanaiuaninnans 7 Step 1 Magnesium and Betaine GONnCEIMIANON ssecsccisciiecccssdtecetecssesacens tereteceecasteatecetacetacedeeneesneeatenees 7 Step 2 Temperature OptiiZation cccccccccccccsccceencecenceeseeecseneeceaeeseneecseneessaeeseeeeseneseseaseesnssenenees 8 Oer ODUIMIZAUON NOLES ie aa E E A EE e a E E E E E E E E 8 Reaction time and temperature ccesseecescceesseeeeesceeeseeeenseeeeeaeeseaeeesaaeeeeaeeseaeeseaeeesnaeeeeeasessaes 8 Enzyme COnCemiralon sissien eiiiai aaiae Ea aa E aa EE E E EES 8 Primer COnCentration ccccsccceeseeeeseeeceeeeeeeeeesneeceaeeeeaeecsaaeeeeaeeeseaeessaaeesseeeseaeesesesessneeesseeeseaes 8 DDO ixieietee ete eek nana a ected ected eee davies etek eee deed een SE 8 Dilution DUE erisera EEEO EA EDESTEN EEO E EE EN en E ETEEN 8 Additional Amplification Guidelines nsussussensenneunnunnunnunnunnnunnunnennn
13. from to provide a service information or data to a third party in return for a fee or other consideration or 4 resale of the product or its components or derivatives whether or not such product or its components or derivatives are resold for use in research Lucigen Corporation will not assert a claim of infringement against the buyer of this product provided that none of this product or any of its components or any claim in the foregoing patent or patent applications was used in the manufacture of a product for commercial purposes Academic Not for Profit and For Profit institutions must obtain a separate license from Lucigen Corporation to use this product for any purpose other than those permitted above 13 SP003 Rev A
14. ge of temperatures For most targets the optimal reaction temperature is between 66 C and 72 C Reaction temperatures above 72 C or below 66 C are not recommended Note Higher reaction temperatures generally provide faster amplification but may also result in increased background non specific amplification Other optimization notes Reaction time and temperature LAMP and other isothermal amplification processes are prone to spurious amplification if the reactions are allowed to proceed for too long or if they are run at too high a temperature Therefore during optimization it may be necessary to reduce time and temperature from apparently optimal conditions in order to avoid unwanted background amplification or decreased specificity Reaction times of 30 minutes or less are strongly recommended This is true of reactions with the target present as well as of no template controls Enzyme Concentration As with time and temperature the use of more enzyme may result in better amplification results Using the enzyme at up to 2X concentration may increase the reaction speed or sensitivity however it can also lead to increased background amplification Primer Concentration Depending on the primer template system it may be necessary to optimize primer concentration after the optimum reaction condition has been identified Certain primer systems may be prone to background amplification at or near the commonly used LAMP primer concentrati
15. icated to the success and satisfaction of our customers Our products are tested to assure they perform as specified when used according to our recommendations It is imperative that the reagents supplied by the user especially the RNA specimens to be amplified are of the highest quality Please follow the instructions carefully and contact our technical service representatives if additional information is necessary We encourage you to contact us with your comments regarding the performance of our products in your applications Lucigen Technical Support Email techserv lucigen com Phone 888 575 9695 SP003 Rev A Product Guarantee Lucigen guarantees that this product will perform as specified for one year from the date of shipment Please avoid using reagents for greater than one year from receipt Lucigen does not guarantee the performance of the the QIAGEN Lake Constance solutions For technical questions on the the QIAGEN Lake Constance products contact QIAGEN Lake Constance Technical Support service qic giagen com For information on QIAGEN Lake Constance solutions visit www giagen com ESE Product Description The OmniAmp RNA and DNA LAMP Kit is intended to simplify development of LAMP reactions for detecting RNA or DNA LAMP is a commonly used isothermal amplification system that was developed and patented by Eiken Chemical Co see Appendix D This kit may be used for research purposes only under the limited use license de
16. nnnunnnnnunnunnnnnnnnnnnnnnnnnnnnnnnnnnnn nanna 9 Avoid Ribonuclease RNAse COntarminatiOn 22 cccccccccsccceeneceseseeeeneecseneeeseaeeesaeeeseneeeneaeeesneeenenesens 9 Cold REACTION S t Up o PEPEE 9 Template Preparation nccccnncn nn nine nine ne eee ee 9 Reaction Overlay 3 sees carated seed ccel ak ceed aiaa n aa eh ataccnebeacsanehseanceal asksaakcccnceaecg AEEA sastenarsaamebeneniues 9 ONTE Se e A A E A cagesuceecseccan tvececcctiaceesds 9 Dy y dor guantitati Osennii AA AEE ALTELE nde AAL ALAALA EAA 9 R ferenNtE Siae aaa daaa aaa aaae aaea aaa aa aa Aaaa Aaa adaa dadada adaa ddaa aaas 9 Qiagen Workflow Information ciccecesceccesccesecciesssesesesessdsseessnsscetesciesedusoissesnesdessevieseieisceseiadereesdutsceiacess 10 APPCN ICES cseeeeeeeeeeeeeneeeeeeeeneeeeeneneeeseeeenseeeeneneeaseeeegseeeeseeeeaseneeaseeeeeeneeaseeeeaseeeeneneessenseaseeeesseeeeseneenes 11 Appendix A Examples of Target Specific and Nonspecific LAMP Amplification 1 c 0ccccccc 11 Appendix B LAMP Resources csccccceetseeeesenneeeeeeeaeeeecseaaeeecseaaeeseseeaeeeesseaaeeeesaaeesesseaaeseeseaeeeetes 11 Appendix C Quality Control ASSayYS es as1aeineeeennnenneeneeneenneeneenernnnnnrnneenennennnnaneinennennneaneenennnnnneene 12 Appendix D Notice of Limited Label License Copyright Patents Warranties Disclaimers and Trademarks cresen a EE EE esse eet EN anihan ra STE ENRERE EEE E a EN E E ERE ESTA 13 Technical Support Lucigen is ded
17. ons If undesired background amplification is observed a primer concentration titration down to 0 2X of the original primer concentration should be performed The concentration of all primers should be adjusted in unison preferably by using varying amounts of a stock of the primer mix Increasing primer concentration will generally lead to increased background amplification and is therefore not recommended Reducing the primer concentration may reduce sensitivity and reaction yield or it may increase the time required to amplify your target Dye It is recommended to use EvaGreen dye however users are free to use any fluorescent dye suitable for use in real time PCR However in such case optimization to determine required concentration of dye to be used in LAMP reaction will be needed Dilution buffer Preparation of target dilutions in 25 mM Tris pH 8 0 usually helps in increasing sensitivity especially for RNA targets When using Tris as dilution buffer it is very important to adjust pH to 8 0 0 1 and filter the solution Prepared solution can be stored at room temperature however for long term storage and to avoid contamination it is recommended that solution should be aliqouted and stored at 4 C SP003 Rev A Additional Amplification Guidelines Avoid Ribonuclease RNase Contamination Major sources of RNase contamination in a typical laboratory include solutions and reagents environmental exposure and contact with hum
18. particular application in which the product is used It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties Limited Label License The OmniAmp DNA Polymerases is covered by patents assigned to Lucigen Corporation Patent Applications WO 00 28082 WO 01 34790 and WO 01 77317 regarding the LAMP method are owned by the Eiken Chemical Co Ltd OmniAmp is sold by Lucigen under license for use in LAMP for research use only Lucigen does not encourage or support the unauthorized or unlicensed use of third party intellectual property It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties If the purchaser is not willing to accept these use limitations Lucigen Corporation is willing to accept return of the product for a full refund For information on obtaining a license to use this product for purposes other than those permitted above contact Lucigen Corporation 2905 Parmenter St Middleton WI 53562 Email Lucigen lucigen com Phone 608 831 9011 Fax 608 831 9012 The consideration paid for this product grants a Limited License to use the product pursuant to the terms set forth in this Limited Label License Academic Not for Profit and For Profit institutions acquire certain limited nontransferable rights with the purchase of this product see below By use of this product you accept the terms and
19. scribed at the end of this document Details of the LAMP reaction and its use can be found in the References section or Appendix B LAMP Resources OmniAmp DNA polymerase is unique in having both reverse transcription and strand displacing activities This rare and powerful combination enables LAMP detection of either DNA or RNA targets Other isothermal amplification techniques that rely on strand displacement may also be possible with OmniAmp reagents however they are not tested LAMP commonly employs a set of six primers which must be supplied by the user Lucigen recommends using previously established designs or designing new primer sets using the Eiken web utility see Appendix B LAMP Resources Not all primer sets identified by this program are guaranteed to perform with OmniAmp or any other enzyme system It is recommended that two or three primer sets be designed and compared experimentally We highly recommend inclusion of loop primers Nagamine 2002 LAMP amplification may be detected by agarose gel electrophoresis turbidity Mori 2001 or by using double stranded DNA binding fluorescent dyes Product Designations and Kit Components Product Kit Size Catalog Reagent Description Part Volume number Numbers OmniAmp DNA Polymerase 50X Peat PDHL 10X DNA Polymerase Buffer C F881958 1 1 mL Magnesium Sulfate 7 OmniAmp Be che 30065 1 100 mM Sucked ae RNA amp DNA Betaine 5 M F
20. ual for this software has been published online as well PrimerExplorer link http primerexplorer jp elamp4 0 0 index htm Primer Explorer manual pages 1 through 10 https primerexplorer jp e v4_manual pdf PrimerExplorerV4_ Manual_1 pdf Primer Explorer manual pages 11 through 34 https primerexplorer jp e v4_manual pdf PrimerExplorerV4 Manual 2 pdf Primer Explorer manual pages 35 through 68 https primerexplorer jp e v4_manual pdf PrimerExplorerV4 Manual 3 pdf Appendix C Quality Control Assays Activity Assay Polymerase activity is assayed at 72 C with 0 2 mM each of dATP dGTP dTTP dCTP mix of unlabeled and P dCTP 10 ug activated calf thymus DNA and 0 1 mg mL BSA Absence of Endonuclease OmniAmp Polymerase is determined to be free of detectable endonuclease or nicking activity One ug of supercoiled plasmid DNA is incubated with enzyme for 16 hours at 70 C Agarose gel electrophoresis shows no alteration in mobility consistent with endonuclease or nicking activity Absence of Exonuclease OmniAmp Polymerase is tested to be free of contaminating exonuclease activity by incubating 1 ug of Hind IIl digested lambda DNA with enzyme at 70 C for 16 hours Agarose gel electrophoresis shows no alteration in mobility consistent with exonuclease activity Absence of Ribonuclease OmniAmp Polymerase is tested to be free of contaminating RNAse activity by incubating with a fluorogenic RNAse substrate for 1 hour at 37
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