OmniAmp™ RNA & DNA LAMP Kit
Contents
1. 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 at 2 mM final concentration However certain targets and amplification systems will require optimization using the included Magnesium Sulfate and Betaine supplements For most targets optimization of Magnesium and Betaine concentrations will result in shorter time to result and reduced background amplification Experimental Reaction Final Concentration or Nuclease free H2O 10X DNA Polymerase Buffer C 100 mM MgSO Target Specific Primer Mix OmniAmp DNA Polymerase 50X Template RNA or DNA 1 Please see the Primer Design section of this user manual 2 For ease of handling the Betaine solution may be diluted to 1 M in nuclease free water 3 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 4 Buffer C is prepared with low magnesium 2 mM final to allow optimization However most LAMP systems will require a final MgSO concentration of 6 mM or greater Note If you plan to include additional reagents2. 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 e Target RNA or DNA MA148 Rev B OmniAmp RNA amp DNA LAMP Kit 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 Make sure that thermocycler or heat block is set to 70 C Note that when using a heat block it is recommended to use 0 2 mL vials and to monitor the temperature closely If using larger reaction vessels use a water bath or overlay the reactions with mineral oil RNase free environment and procedures should be used to avoid contamination Add target in an area separated from the area where the reaction mix is prepared Thaw reagents and set up reactions on ice 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 in order to better familiarize themselves with LAMP and the OmniAmp system Control Reaction NOOR Table 1 Control Reaction Setup Dee a E E C dT SCC ses iesmmoen S om a8 LAMP RNA Cono Primer me Tox xO Omniamp DNA Polymerase 50x x 15 Dion of RNA Cora Taavon Ooo S S 1 RNA Control can be used at dilutions from 1 5 to 1 500 with reliable results Wor
3. 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 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 MA148 Rev B OmniAmp RNA amp DNA LAMP Kit Appendix C 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 resp
4. 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 A 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 Product Kit Size Catalog number Product OOOO 100 Reactions 30065 1 TM OmniAmp RNA amp DNA LAMP Kit 500 Reactions 30065 2 Components and Storage Store all kits and components at 20 C ye max 25 C min 1 OmniAmp RNA amp DNA LAMP Kit consists of the following components Description Part Number 100 Reactions 500 Reactions OmniAmp DNA Polymerase 50X F831942 1 10X DNA Polymerase Buffer C F881958 1 Magnesium Sulfate 100 mM F98695 1 Betaine 5 M F881901 1 RNA Control LAMP Primer Mix 10X F812344 1 Nuclease free Water F98698 1 RNA Control F92697 1 Material to be Supplied by the User e dNTP mix 25 mM each e Target specific
5. such as those needed for quantitation of the reaction product reduce the amount of nuclease free water used accordingly It is recommended to prepare a reaction mix 10 greater than required number of reactions to account for overage Note If a dye is added for amplification quantitation use between 0 2 uL and 0 5 uL per reaction or other per the manufacturer s recommendations Excessive amounts of dye will interfere with or inhibit the reaction MA148 Rev B OmniAmp RNA amp DNA LAMP Kit Isothermal Amplification Conditions The following general program is recommended Step Temperature Time 1 Amplification 66 C 72 C 20 35 Minutes 2 Hold 4C o0 1 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 LAMP Reaction Optimization Lucigen recommends optimizing reaction conditions in two separate steps In the first step determine the optimal Magnesium and Betaine concentrations Then use that buffer formulation 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 perf
6. 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 Thank you Lucigen Technical Support Email techserv lucigen com Phone 888 575 9695 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 MA148 Rev B OmniAmp RNA amp DNA LAMP Kit 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 C This kit may be used for research purposes only under the limited use license described at the end of this document Details of the LAMP reaction and its use can be found in the References section or Appendix A 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
7. Ne Lucigen Simplifying Genomics OmniAmp RNA amp DNA LAMP Kit Please read carefully and thoroughly before beginning IMPORTANT 20 C Storage Required Immediately Upon Receipt Lucigen Corporation 2905 Parmenter St Middleton WI 53562 USA Toll Free 888 575 9695 608 831 9011 FAX 608 831 9012 lucigen lucigen com www lucigen com FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE MA148 Rev B OmniAmp RNA amp DNA LAMP Kit Table of Contents Product DeCTription cccccececeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees Components and Storage iseccccssetisceceeldecies esate deeded ceded Reaction SEU seceuctancutneatnestccerehenewenchendhesetcensercganncheniceoenemenebineuieds LAMP Reaction Optimization 20 00 00 cccceeeeeccseecceeeeeeeeeeeeesseneeeeeeeees Additional Amplification Guidelines cccceeeeeeeeeeeteeeeeeeeeeeeeeees PRCT OR CINCOS os occessis cocpeceaeceed cicasucnden totedouccutecoudenns Loteagtaaguesocespieeetadgucuce Appendices saat eotetoicenndedistodd ant dedioemndeahiedataddedinasnbedeeaetanboeeniebbaiied Appendix A LAMP R SOUICEG sssssssssssssnsssssssssnsssssneanees Appendix B Quality Control ASSAYS 22 c csseeeceeeeeeeeeetetnteeeeeeeens Appendix C L gal siasacahosossrasceteretes yi dpese sree A AR reas tnadiosediniaties Technical Support Lucigen is dedicated to the success and satisfaction of our customers Our products are tested
8. ect 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 the 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 partie
9. er 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 therefrom 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 11 MA148 Rev B
10. inent primer dimer band is also characteristic of non specific amplification Lane 4 Negative LAMP reaction MA148 Rev B OmniAmp RNA amp DNA LAMP Kit Additional Amplification Guidelines 1 Avoid Ribonuclease RNAse Contamination Major sources of RNAse contamination in a typical laboratory include solutions and reagents environmental exposure and contact with human hands and skin Avoid introducing RNAses rather than trying to remove them Some basic precautions must be taken to work successfully with RNA Always wear gloves to prevent introducing RNAse contamination from human hands Change gloves frequently especially after touching skin door knobs and common surfaces Use a set of pipettors dedicated solely for RNA work Use RNAse free plasticware and reagents Designate an RNAse free area of the lab 2 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 65 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 3 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
11. 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 4 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 be overlaid with one half reaction volume of PCR grade mineral oil This may slow the reaction 5 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 6 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 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 pyrophospha
12. kflow 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 Thaw all kit components and hold on ice 2 All components should be mixed well before use Vortex and 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 RNA Control I 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 22 5 uL of reaction mix in a PCR tube or 96 well PCR plate for each reaction 6 On ice prepare 1 5 dilution of RNA Control by combining 1 uL of RNA Control with 4 uL of Nuclease free Water 7 Add 2 5 uL of the 1 5 diluted RNA Control I 8 Cap tubes or seal plate wells Centrifuge briefly to collect prior to incubation 9 Incubate at 70 C for 30 minutes 10 Immediately stop amplification reactions using one of the three methods below This step required to stop the enzyme activity a Hold on ice or at 4 C MA148 Rev B OmniAmp RNA amp DNA LAMP Kit b Add gel loading dye with 50 mM EDTA c Perform a heat kill step in a thermocycler or heat block at 95 C for 2 minutes 11 Run samples on a 2 agarose gel Note Reactions may be kept at
13. orm all reactions at 68 C A Adjust the final Magnesium Sulfate concentration in 2 mM increments Final concentrations of between 4 mM and 10 mM are often optimal and can be achieved by supplementing the reaction with 100 mM MgSQ solution In a 25 uL solution each additional 0 5 uL of 100 mM MgSO solution increases the MgSO concentration by 2 mM Note Polymerase Buffer C already contains Magnesium Sulfate at 2 mM which is not sufficient for most reactions Therefore most reactions will require supplemental magnesium sulfate Example Use 1 uL of 100 mM MgSO in each 25 uL reaction to increase the Magnesium Sulfate concentration in the reaction from 2 mM to 6 mM B Adjust the final Betaine concentration in 0 2 M increments Final concentrations of between 0 1 M and 0 7 M are usually optimal however some primer template systems work best at up to 1 0 M In a 25 uL solution each additional 0 5 uL of 5M Betaine increases the concentration by 0 1 M 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 Example Use 2 5 uL of 5 M Betaine in each 25 uL reaction to reach a final concentration of 0 5 M Betaine 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 Note The use of buffers o
14. r 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 concentrations 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 MA148 Rev B OmniAmp RNA amp DNA LAMP Kit Examples Agarose Gels of Target Specific and Nonspecific LAMP Amplification Correct target specific amplification Spurious background amplification Figure 1 Lane 1 100 bp Marker Lane 2 Negative Figure 2 Lane 1 100 bp ladder Lane 2 and Lane 3 LAMP reaction Lane 3 and Lane 4 Positive LAMP Background Amplification in a LAMP reaction Non reaction products from an RNA Control template A specific or Background amplification appears as a distinct banding pattern is seen among the smear single continuum of fragments with no visible or indistinct bands A prom
15. s 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 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 buy
16. te 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 MA148 Rev B OmniAmp RNA amp DNA LAMP Kit 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 82 Appendices Appendix A LAMP Resources 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 manual for this software has been published online as well PrimerExplorer link http primerexplorer jp elamp3 0 0 index html Primer Explorer manual pages 1 through 16 http primerexplorer jp e v3_manual pdf PrimerExplorerV3_Manual_1 pdf Primer Explorer manual pages 17 through 37 http primerexplorer jp e v3_manual pdf PrimerExplorerV3_ Manual 2 pdf Primer Explorer manual pages 38 through 37 http primerexplorer jp e v3_manual pdf PrimerExplorerV3_ Manual _3 pdf Appendix B 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
17. ther than Buffer C provided in this kit is not recommended MA148 Rev B OmniAmp RNA amp DNA LAMP Kit 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 range of temperatures For most targets the optimal reaction temperature is between 66 C and 72 C Reaction temperatures above 72 C or below 64 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 Prime
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