DNA増幅試薬キット
Contents
1. AL CBB FiSERMOS ER CETIS P 6 HILAR S 4 EAO primer amp SAS SCOBRREMSL O SIMEON S lt MRMICISIBTRE CHS O SIRE EDZ BARWICHLTIS gt SORMSSI SM UEBTISIBATS A y bla LAMP AERTS Fy b DNA ASB StI EONAR Y F T DCYAIIVCE COMBE ERE 60 65 BRIL63 C CRS ER GR C1 BS RAIDET DNA OLIN CEST 9 be 1 2x Reaction Mix RM 487A 967AKD 192742 0 6mL 1tube 2tubes 4 tubes 2 Bst DNA Polymerase Bst DNA Polymerase 3 Distilled Water DW 4 Primer Mix DNA PM DNA 5 Positive Control DNA PC DNA 0 1 mL X1 C Ald FA DICECHAN THISRMNTSI x2 R 2 x Tris HCI pH8 8 KCI MgSO CNH4 2504 Tween20 Betaine dNTPs sree 60uL 1tube 2tubes 4 tubes 1 0mL Iltube 2tubes 2 tubes 25uL 1 tube 1 tube _ oa 40 mM 20 mM 16 mM 20 mM 0 2 1 6 M 2 8 mM each X3 Positive Control DNAld ADP YD NA ZAIRE Hind CHILL FO BR 6 557 bp ADASRICMAIAATEOTS X4 96 FZ KDE 192 FAbDICIS Primer Mix DNA RU Positive Control DNA SSENTHIVEA CAE RE LAMP id 4 EO primer CHS HSM AID DNA Polymerase ATRE FORRES RRBISIBATS 4 MO primer D55 2 XO inner primer dt ED 3 GUC 5 BU CHABAD ORCS 2 IAs TS primer T 5S MOBBUIL ED 3 ADS OBR BU CSA UIE 1B FH BR BA Sh AICP ILI SKIRELET Stemi CO inner primer CHOFRMISAZTAIL TBiIEP SORCHRERM C IL FERITA PILLE in2. TAB UIE ORES lt DREMHOES 2 RMF rA Tid BIR LOT WOT BO ABEDE MWIRUUCIAERUT CIES ROF a Did A SaglIc ERSDRUCCE Fai TER ELSOHSCELK METSAUIIMNOD KORESSRISTRMMHDOLT Loopamp SEE WA CHEBBUT lt SIESU Rit Fa TORBIC WERE CUPILATL DYER Yb ORAZ AREL RATRE IEDR 4 ISIE RMICRL COB RM VRZS SVIRZCPY IW SREB RIMA RAN o THISCBERED YINTCFA TES ROMEOR 4 RMF A DICVAI SVIA GYTIVEROADIEN TSCCA Stee Cia LT lt IERU TORR LicinGld RIDRD RBA CERNERET EnF MIRCBORHEOR CEVETOC WAM LEURS RUTES Rad DD TWSWEICIA AEVIDY UT MIG SEN BRUT lt IERU 5 SARORMF a FORIR 1 RMBOF I P REPSROY CROWLT lt IESU HHRADISIBE WICKS iB R DRATI CIERSBISDY CE lt RARATON EN EERU E ULARO DNE SSTAEEMS OES 2 RIMROF 7E vy PARIS IC RAEI THL REDRE o TRUT ESN RICA KIL PMUBISINBUVT lt CIES R USA E ISRO EOE 1 LAMP RIGISIERICRBVERIOG CHO if TSCROEBRESESIR Ya VEORI SED ABRUVY DI ILOBLIA TA a 3 BU Id CASE Whi Yb XIRRI CIRO Bei C amp SE_ DRE o ISIE MORAUBILDIED RRO PF ERICFA JOF Y y IDBDBUES YDER a VISWED BRERA LBRO blk BEMSO DNA AC lt MBCEBA DHVES COKIBIVISR EBRODU YNYFSE THBRRORUWA CHSBERRY CUPI
3. 1 Notomi T et al Nucleic Acids Research 28 No 12 e63 2000 2 Nagamine K et al 3 Mori Y et al Clin Chem 47 No 9 1742 1743 2001 Biochem Biophys Res Commun 289 No 1 150 154 2001 DS BA th 730 5 amp amp AEICFBABHRWRE 2000 S ie 230 ADFEMF A FAIOISA ABSR 2000 6 8 moh th 33 26 SEAT TIL 7 agamine K et al ADFLEMS 8 BAMIBFA N1ARTIT4 BAB AWS Nes RE SRA B ARAB ENR DU A 4 7 U BI 0120 308 421 RE AR TT IT A 2 2 X pal mBLSs 2003 Molecular and Cellular Probes 16 No 3 223 229 2002 Be ea 54 No 3 667 715 1999 KME FRARI HARTER BSKS 43 Read his instruction carefully before use For research use only Rev January 2008 Ver 4 4 Loopamp LAMP Loop mediated Isothermal Amplification method DNA Amplification Kit Characteristics amp enzy cond 4 pri cation efficiency and enables amplification within a prod detection possible This kit is a simple reagent kit for the amplification o LAMP Loop mediated Isothermal Amplification method is a novel gene ification method capturing the following characteristics Only one me is required and the amplific
4. ARE VERS TSAS ECI F4 Loopamp 36 BRRUWARORBSS CE 4 BAAD CORY SIBEMICKSEDYVSISR Yavieta RIESEN VEROL REET OC lt IESU FIILI ISIESNcaS RDR O 5 2 ul AT 2 FBEOPHAO YT ECEtBr HSlv kt SYBR Green I TELET I LAMP RM K StS MA ISS ORO IBLOS CBAAMSTNET TESIER EREOMA CHSCEDS CR LOBES Ts 1 2 CTE buffer amp EORR AF y bAWS 20C CREULTCIESU TSaURICIT 2D SRO Y IERTA REOLER ACE EE HEROTE DICT TCIESUN ROR RISER CI PRCRFIDIKE CTD TCE ARSE BALEYIDYUTFlA FOS SOF y y TICNSU CW SMEBABEL BZBEYIDYUTPSCEA lt SESU otk ER tORGUBE BIEDS YASS YERLI ROA ILEBILD EEH EAS HUCCEA lt IERU CRHBARERS 20 DRORU TROREAH RATRI TIR Bst DNA ETAN Polymerase SRIET SENMOILT OC MUHULE IES RABRREHNBENB Am DUTIES ROMRICIL EDTA 0R BHL hEm SY D buffer VTS ESL BBFU MESMARMBICASCVYAY TAYBAL FhSN BHOBBICBAR lt BHERLET EEIT Ca Zn Fe 1AY SOR BALTES 3 RMF A TFORIRUAE 1 3 Saw st at AVeazseal SORS6SRVEOR RW OBSIA CEO ETOT RGF A TIAWFSAAD Loopamp RMF a FECAL SEW MNO RMF a
5. YPWAT SABERE AURE OLYTIA Eiken GENOME SITE URL http loopamp PA lt CIESU FE REFORMI REOARNRKAE ATSR 2 TY RRT Y MARRE Loopamp IY FWT Y PARRER E E 3 R LES DES SED PNR CRY ROTES 1 2 SHER zo eiken co ae ECE LSL JPII T LBGOTE NSTET Ree BUSCEG LYERT Y KTORE RUOCSET IREO OWS CEIR lt IESU BB LY RAT Y KARB ARESE CORICISERISH OA RIRE BIED Loopamp Y te PURHAREBISCOT SIB RMR TR 3 OMRIRH Be RHETT E CRS 240 260nm 350 370nm A LERS CHRBULET Batt BIY k WRH 320nm MENDRE WF 2A PRBKO MRR UL CRMF A FORMBKO ARRE CRE IVCRICREDRHAR I NISE Pay IVEBRICER ER UII CHELES IM CEBHARUTRASCECDHVIETON zE ILER U THEUTSIESL KE KIR R OM patty O U tE RA RBOWD NSA aS lahat YVhO IVBRHERUCRASCOMHVIE SM Zo HLEV Fa FORES E ASECUT BIY k SF I DPERINRRNREDS behave ny bO ILO D ADEN FTSKIICL RUTCKIESU fy Fan 4 lk Loopam p Re RE UPILITA LYRATY A O ED HARD YFAN S CORREN 0 5 CAUA hy KRY RY FD BRBUSCCMCET SB BH BRR H
6. angle of the reaction tube so that the difference between positive and negative controls becomes observable The incubation can be carried out in the Loopamp Turbidimeters Realtime or End Point or in the commercially available incubators required temperature accuracy within 0 5 C with hot bonnet Turbidity detection is also compatible with visual florescence detection using florescent reagents For more information refer to the package insert for Loopamp Florescent Detection Reagent Electrophoresis In order to avoid contamination extra care should be taken when handling the amplification products during electrophoresis process 0 5 2uL of the reaction solution is applied for electrophoresis with the 2 agarose gel The gel is stained with ethidium bromide CEtBr or SYBR Green I A ladder pattern can be observed in electrophoresis since the amplified products consist of various size of inverted repeats of the target sequence on the same strands VS VY Caution for use 1 1 Handling the kit This reagent kit should be stored at 20 C To prevent the reagents from deterioration only take out the necessary amount of reagents from the freezer before use No decline was observed in the kit performance even after repeated freezing and thawing for 20 times in the quality control testing But in order to maintain the reagents performance keep off unnecessary freezing and thawing 2 Thaw the reagents at ro
7. 3 Detection x In order to prevent contamination the following procedure 1 or 2 is recommended for detection and data recording which enables the detection in a closed tube 1 Real time turbidity detection Real time turbidity detection can be carried out with the Loopamp Realtime Turbidimeter For information about the equipment visit the Eiken GENOME SITE CURL http loopamp eiken co jp e For the detailed operation of the equipment refer to the instruction manual of the equipment 2 End point turbidity detection B 4 End point turbidity detection can be conducted with the Loopamp End Point Turbidimeter For more information refer to the instruction manual Notice There is no relationship between the end point turbidity values and the initial amount of the template Visual florescence detection Visual florescence detection can be achieved by using the Loopamp Florescent Detection Reagent available for sale separately UV lamp wavelength at 240 260nm or 350 370nm protective goggle or glass board are required for florescence detection When UV lamp of wavelength around 230nm is used negative sample may look like radiating fluorescence Judgment should be done by comparing fluorescence of sample with that of positive and negative controls When output of UV lamp is too strong negative control may look like radiating fluorescence In such a case take the UV lamp away from the reaction tube or change the
8. 43 Nogi Nogi machi Shimotsuga gun Tochigi Japan Manufacturer Rikon lt ta RRO O RMFA TPAAAP SRS yD O X OovyaPtAa RUPTARYIA O Meh ARs O 8 7V1 2707F 7AA D RMY O MILF YyDIASHET lt UPILST LBBRWORS O Loopamp UPILIT ASEAERS lt DY ERT Y PSERWORS O Loopamp LY RRT Y HSERESS gt SD Ob O Loopamp BRRR O Loopamp UPILAT ABERNERE Loopamp TY RRT Y HSER RIE REI LATYNS CARRE O0 5 CAA Ry KRY RY MY O k 70y9 CBB LEAR 2 ERM IRGA SROVAY 1 Loopamp RMFA Alc CYAIRMBCDY K SyI7AZ23uL DiELES 2 DY TIVISW 2 uL BRDU 25ulL clas KEDYE JLERMFC UT d BIY FO vIcle Distilled Water DW 2u L BIY FKO vIcld Positive Control DNA PC DNA 2uL ALAD COCR ENyF1 VD Mit vy TAROEETOS VEVTICKOR lt IBS LIER ZEVIDYLE F KE RAORARVAMWERWKDICSEBLES 1 Loopamp BENERE CUPILSTA LYRNT Yb Rdbt yFan I CRERBEDO 05 CUA Ry bRYURY bY CHR DERSORM Fa Deeyvbu 60 65TC 63 C T 30 60 DET YFAN FL KS Et LIE primer CRo CRHMBRSOC BSD CORHFRIOVES CS 2 80 C 5 DMIF 95 C 2 DMT YFAN FISCECICKY ERARE RMABLACES 172 3 RE x DYPSR YVave d UPILAT LBS e
9. I SyIAZBRARMAF a TICSWAREWERT ALBA lt at gt lt As gt 2 x Reaction Mix RM 12 5 wl Primer FIP 40 pmo BIP 40 pmo Loop F 20 pmo Loop B 20 pmo F3 5 pmo B3 5 pmo Bst DNA Polymerase 1 0 uL Distilled Water DW X ul S 6 at 23 0 uL F7Zk x6 WSF ULSBMBHO EAD Loop primer ANS CC TISERE 1 3 Ic menk O IY KO ILRA lt at gt lt As gt 2 x Reaction Mix RM 2 5 ul Primer Mix DNA PM DNA 2 5 ul Bst DNA Polymerase O wl Distilled Water DW 7 0 wl 6 at 23 0 ULI TAF Z SBRRWOSIS bieVAZI SyIRZICHFEO Loopamp Bt BRR MRE lul MDL K I 23 uL ELUTEE x x J31 Eey kCMAHENETEAT SBS YVAZI SyIAZORRSSTIS TX EY FORRICH TLIESL 2 DEB FA PeB lt ROMNCRSEIS OAF Pye yYVeMS D RISA HSVUANLFy IASF D ICT 1 WAX GORRICKO D 4 8 ROLIEK MESDAUBICAMOITT GURY AS SyI2ZCLA KET STEMS IETOT AZAEYSOYVCHS Che ES WUT yy DASFU COPD RIC SC BRO pax De Lie EZ ZI ZSyIAST CICA UT CIES BUIUT lt KIESU BH FZ 1 VZS S9PREVYINERORS KETHIST lt SESU 7 Nagamine K et al Molecular and Cellular Probes 16 No 3 223 229 2002 8 The guideline for the bio safety and bio hazard by the Japanese Society for Bacteriology Japanese Journal of Bacteriology 54 No 3 667 715 1999 Licensed under U S Patent 5 814 506 EIKEN CHEMICAL CO LTD 1
10. LSTA IYF HU CIID TES HE RUD YFIR EIZIE BK SI B ABH ICWETERKARCLE KERMIIPDDNISY FERSUVEMHSC DFi 70AT TSB EMEARI BTI TLEN HB RHER CMBR RE SAS D8 SEET MIPDSY IS RBER OMIT TEGET LTO KIRA BH Bold she DUT EO RMA SNSARIME ICANSC EIS DYB lt TE ITEE XIIEN CHELTCIESU 3 BRUODSRAZRIMS TARER RURUVCDUY CISMBIS V1 A V RIRE oT 4 Loopamp RAF 7 YZI YI ZR UV RHICKSES BBSCRIEBRECESTE BIDAIC TIEFA lt ER BG BEBROWESRSSCRAEDHORT BREE LIERHEORSO TLEN 5 AFy hid FWAR 6 BEF RAOAG OR OQ FEY KORACH o Cita FRAO Aes BRCRASRIBUT KFY KOREI HE KCEOHE tA 8 ERE GICISU lt IESU RUE ICBRUCHE Lic d WSADSARABID VEO ARS PREF 2a TDICIAUV RA UU BmODHIES B GRF AER BES IChSE RoR ACUT 8 SMBICRMOBABABR Exp Date WICIBRRUT lt IESU HRF J7APP FESS IC BS SMERV UV CMUBUT lt IES y I Z DREN ITEUDA BMRB RDF PO s EMACUTWEAT BERORGER KE SBSLASOSMIRAMICHL Bi ORCS Ras aR FADA SWRA RI F A8 FAK LMP204 Loopamp DNA 8 h 96 FAR 20 C 1 LMP205 192 FAKD LMP206 23x
11. ation reaction proceeds under isothermal ition It has extremely high specificity because of the use of mers recognizing 6 distinct regions on the target It has high amplifi shorter time It uces tremendous amount of amplified products which makes simple DNA by the LAMP method and amplification of target DNA can be conducted by simply incubating specimen and all reagents provided at a constant temperature 60 65 Contents of the kit 1 2 x Reaction Mix RM 2 Bst DNA Polymerase Bst DNA Polymerase C for a fixed period of time 1 hr for standard 48 tests 96 tests 192 tests 0 6mL 1tube 2tubes 4 tubes TE PAE AEEA AT FELTA ITTE T ATT 60u L 1 tube 2 tubes 4 tubes 3 Distilled Water DW 1 0mL 1tube 2tubes 2 tubes 4 Primer Mix DNA PM DNA 25uL 1 tube 5 Positive Control DNA 3 4 PC DNA 0 1 mL ltube 1 2 3 The control DNA is a plasmid DNA that is inserted with a Hind The notation on each reagent tube is shown in composition 2 x Tris HCI pH8 8 40 mM KCI 20 mM MgSO 16 mM NH4 2804 20 mM Tween20 0 2 Betaine 1 6 M dNTPs 2 8 mM each fragment 6 557 bp of lambda phage DNA 4 Primer Mix DNA and Positive Control DNA are not included in the 96 Pri tests and 192 tests package nciple LAMP method is a novel isothermal nucleic acid amplification method using 4 kinds of primers and DNA polymerase with stra
12. ection gt O Loopamp Fluorescent Detection Reagent O Loopamp Realtime Turbidimeter gt Loopamp End Point Turbidimeter 5 or incubator with hot bonnet temperature accuracy within 0 5 C O Heat block for termination of the reaction 5 O UV lamp wavelength at 240 260nm or 350 370nm O Protective goggle or glass board 5 For the information about applicable instrument reaction termination unction and condition for UV irradiation refer to Eiken GENOME SITE CURL http loopamp eiken co jp e 2 Primer design Appropriate primer design is essential for amplification by LAMP Use exclusive primer designing software for the primer design Refer to LAMP primer designing software PrimerExplorer at the website http primerexplorer jp e Using the highly purified primers a rapid gene amplification can be performed and stable reproducibility of the amplification can be obtained The first screening for appropriate LAMP primers might not necessarily require highly purified primers However after the primers are determined it is recommended to use purified FIP and BIP through HPLC or better purification 3 Reagents preparation 1 Take out the reagents stored at 20 C and thaw them at room temperature Once the reagents are thawed keep them on ice 2 Preparation of master mix Operate on ice 1 The following amount of the component is required for one reaction O Sample reaction lt Reagents gt l
13. ix and sample solution Operate on ice 1 Dispense 23 uL of the master mix into each Loopamp Reaction Tube available for sale separately 2 Add 2uL of sample DNA to the master mix and the volume of the solution should be 25yL in total For control reactions use 2uL of Distilled Water DW for negative control and 2 uL of Positive Control DNA PC DNA for positive control Mix the solution well by pipetting or tapping the tube with the cap closed and then spin down Be careful not to cause air bubbles when mixing 2 Amplification reaction 1 Set the reaction tubes in Loopamp Turbidimeter Realtime or End Point or the incubator with hot bonnet temperature accuracy within 0 5 C and incubate them at 60 65 C for 30 60 minutes The reaction condition is dependent upon the characteristics of the primer used therefore examine the optimum condition beforehand Inactivate the polymerase and terminate the reaction by incubating the mixture for 5 minutes at 80 C or 2 minutes at 95 C 2 VU 3LP2019 D
14. lfunctioning By comparing the solution volume in all tubes check visually if proper amount of sample solution master mix has been dispensed into the reaction tube Caution for amplification reaction Since bubbles in the solution will interfere the turbidity measurement and cause false judgment try not to cause any bubble when mixing the master mix and the sample solution If bubbles are present spin down to get rid of the bubbles 2 2 5 Handling reaction tubes after use 1 The caps of the used reaction tubes should not be opened Contamination of amplified products on other samples may not only cause false judgment of the test result but also pollute testing area In this case a correct test result may not be obtained unless pollution is completely removed 2 Keep the cap of the used tube completely closed and dispose it according to the relevant regulations and instructions by incineration or after double bagging it with sealable vinyl bag To prevent the amplified products from dispersing do not conduct autoclave sterilization treatment for disposal Caution for Handling At This kit is designed for research use only Use the kit before the expiration date which is labeled on the outer box The reagent tube is made of polypropylene and the main material for ki LAMP reaction is very sensitive and even the slightest amount of amplified product tainted into the reaction might cause false result The
15. nd displacement activity Among 4 kinds of primers two of them are inner primers whose 3 end diffe repli com end and 5 end are respectively designed to be complementary to two rent regions of the target sequence When the primer linked strand is cated resulting the 3 end structure to self anneals to the plementary region in self structure it forms the loop structure at the This stem loop structure will allow the 3 end to initiate self elongation and another inner primer to anneal to its loop region to synthesize new DNA strand with strand displacement manner Through the repetition of these proc cont esses the amplification proceeds and this enables amplification to inue under isothermal condition with only one kind of enzyme For further details of the LAMP method refer to Eiken GENOME SITE CURL http loopamp eiken co jp e How to use 1 O0O0000000 Materials required but not provided Sterilized tubes for master mix preparation 0 5 mL 1 5 mL Micropipettes 0 5 10 uL 10 100 uL 100 1 000 u L Pipette tips with filter Loopamp Reaction Tube Aluminum rack for cooling tubes Ice crushed ice and ice box Centrifuge for micro tubes Centrifuge for 8 connected tubes Vortex mixer lt For real time turbidity detection gt O Loopamp Realtime Turbidimeter 3 lt For end point turbidity detection gt O Loopamp End Point Turbidimeter lt For visual fluorescence det
16. ner pr imers DSORBRSR RM SROBS CeGETLES CHICKO LAMP AISA Simi ERUETI TSB RRENDA Eiken G eiken co jp ACBaR lt ES UEA A SBR 1 BCHSICEBNSS ENOME SITE CURL http loopamp 1 EBER RB AE Fy HCSENTWECAOT HRBBUTC lt IESU O VAS SyIARRARAF A TD 0 5 mL O EXyk 0 5 10uL 10 100uL 1 O D1tWI MhEFYTD O Loopamp RAF 2 7 I amp 1 5 mL 00 1 000uL O FROMRERN RE CXR 240 260nm 350 37 O DEORE XIE Onm 5 AEWRE RMP LERERE KIMRRAORHZTODLTIA Eiken GENOME SITE http loopamp eiken co jp CSR lt ES 2 Primer M83 LAMP KICK SISIBIC E o CME primer 28t CURL BEATCEROEIO sat SHED TIA LAMP SAO primer Et VEY F BUD Hd Net Laboratory LAMPSA DS V VI KOXP Primer Explorer C http venus netlabora RE primer SROTL RICDUVTIA LAMP 3 amp RIRES SO RMGACRELES tito ZE CHA lt IESL primer a8 DE ory com partner lamp CB8R lt ESL DBE primer OMHREDMSU T primer D RZJ J IZY NCUTERI SREISHBNIABRIL FUE TANIR TACT DY primer RET SRROURERIS DECC FIP BIP CDUVTIA HPLC R TU FICKSSGMEHBLET 3 BRORS 1 20CCREUCIVESHRAE Ea CERU MRADBSICKECREFLES 2 VAS SvyIAZOMR CKRE THO TLES a PI BUEZ FRO IS O FAKON CHELET O FUTILE
17. ny consequential damage derived from the false judgmen caused by non capability problems such as operation error Exp Date case is paper The institution disposing the reagent tube and case should bear the responsibility and abide by the clinical waste disposa regulations water pollution prevention law and any other regulation related Unit Storage Expiration Code No References 1 Notomi T et al Nucleic Acids Research 28 No 12 e63 2000 2 Nagamine K ef a Clin Chem 47 No 9 1742 1743 2001 3 Mori Y et al Biochem Biophys Res Commun 289 No 1 150 154 2001 4 Tomita N et a Abstract for The 73rd Annual Meeting of the Japanese Product Name Unit Storage Expiration Code No A8tests LMP204 Loopamp DNA Amplification Kit 96tests 20 C l year LMP205 192tests LMP206 Biochemical Society 2000 5 Mori Y et al Abstract for the 23rd Annual Meeting of the Molecular Biology Society of Japan 2000 6 Tomita N et a Abstract for the 26th Annual Meeting of the Molecular Biology Society of Japan 2003 3LP2019 D COPRMBBSEL lt iA CO SIERUTKIESU HS 2008 1 AMS 5 4 hk bes 2006 F 4 AWMaT 5 3 hk 4 Loopamp LAMP Loop mediated Isothermal Amplification 3 DNA i8ig a2 Fy F 3 LAMP Loop mediated Isothermal Amplification i amp d O 1 BHOBRBOHA
18. om temperature and keep them on ice for 3 1 2 3 4 gt reagents preparation and later use Before use spin down the tubes to drop down the solution staying on the tube wall or on the cap mix well the solution and spin down again Notice that fierce mixing should be avoided as it can inactivate the Bst DNA Polymerase Caution for visual florescence detection For the preparation of the sample solution do not use the buffers containing chelating reagents such as TE buffer If chelating reagent is added to the reaction solution manganese ion binding with Calcein would be chelated so that the fluorescence light is released even no amplification take place Besides a sample containing a large amount of Ca Zn or Fe ion might cause the false positive test result Handling reaction tubes Only use the specified Loopamp Reaction Tube for turbidity or lorescence detection Other reaction tubes might have different optical ransparency and can cause misjudgment Take full care when handling reaction tubes as they are vulnerable to scratches or damages Check carefully to see if the reaction tubes have any crack or scratch before use Crack or scratch on the tube might not only cause false judgment but also contaminate the equipment If the tubes are broken inside the reaction block of the Loopamp Turbidimeter Realtime or End Point the reaction solution can spill inside the equipment and cause unrecoverable contamination and ma
19. refore avoid this type of contamination and carry out the sample and reagent preparation in different clean benches Conduct the detection of the amplification using turbidimeter for end point or real time detection with which the detection as well as the reaction can be accomplished in a tube with its cap closed Also to avoid the possible contamination from the amplified product when the product is taken out from the tube for the electrophoresis detection Conduct electrophoresis in a room different from that for sample and reagent preparation When ultraviolet lamp is used for the fluorescence visual judgment do not stare directly at the UV light Since UV light is harmful to the eyes even watching for a short period would irritate eyes and cause symptoms similar to conjunctivitis Look at it through glass board or protective goggles When handling the sample always abide by the biohazard counter measures Do not expose the Loopamp Reaction Tube master mix preparation tubes to UV light A change in color or degeneration caused by ultraviolet lamp sometimes results in misjudgment If the operator does not have the experience or knowledge in the field of nucleic acid testing there is a possibility of false judgment Therefore make sure that the kit is used under the supervision of the experienced and knowledgeable technicians Eiken Chemical Co Ltd does not bear any responsibility for false judgment or a
20. t Amount gt 2 x Reaction Mix RM 12 5 uL Primer FIP 40 pmo BIP 40 pmo Loop F 20 pmo Loop B 20 pmo F3 5 pmo B3 5 pmo Bst DNA Polymerase 1 0 uL Distilled Water DW X uL ad qs Total 23 0 uL test 6 Loop primers are not necessarily required However the use of Loop primers shortens the amplification time by about 1 3 O Control reaction lt Reagents gt lt Amount gt 2 x Reaction Mix RM 12 5 uL PrimerMix DNA PM DNA 2 5 ul Bst DNA Polymerase 1 0 uL Distilled Water DW 7 0 wl Total 23 0 uL test x For visual fluorescence detection also add 1 u L of the Loopamp Florescent Detection Reagent available for sale separately and maintain the total mixture amount at 23 u L yx When used in combination with Loopamp Primer Sets follow the preparation instructions of each primer set to prepare the master mix 2 After dispensing gently tap the tubes for a few times hereinafter referred to as tapping or mix the solution by repeatedly inversing the tube or mix by vortex mixer at about 1 secondx3 times After mixing well centrifuge the tubes for a few seconds hereinafter referred to as spin down And the mixture can be used as the master mix for the reaction Notice that too much mixing by the vortex mixer might inactivate the polymerase and assure that vortexing is conducted at 1 secondx3 times The prepared master mix should be used as soon as possible 4 Operation procedure 1 Mixing of master m
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