取扱説明書 - Eiken Genome Site
Contents
1. EAEE z as For research use only Rev January 2008 Ver 4 Pi oaks EREN oo FRG CIR ROTD 1 2 SHE CEU REDES CIMBLTC EU MIBEMORRIILOIED REN 3 peal ma for pana pi hace EOS wo _SMBIGDBUITCIE o amp wavelength a 260nm or 350 370nm UPILAT ANIERE Ped shale esau eels Ke Loopamp O Protective goggle or glass board Loopamp UPILAT ASEWESBERNSCECR UPIT AREDTE ARERIARRUEDIES Se are eas tre aes 5 For the information about applicable instrument reaction termin2. MEBER RE AE Fy HCSENTNEKUAOT BURABUT lt ESU O VAS SyIAZRRABAF A T7 0 5 mL Xid 1 5 mL O EXyk 0 5 10uL 10 100uL 100 1 000uL O D4II NeFyT O Loopamp RAF 2 7 O RMFA TPAAAP SRS YA O X OSyYaP 2Z RUPIZRYIZ O HEADE O 8 7V1 9270F 7AA D RMY O MILF YIZZ H lt UPILST ASRREORBA O Loopamp UPILIT ASEBIERE lt DY ERT Y PSERWORS O Loopamp LY FWT Y HEBRERE lt RHBRRHOB8 gt O Loopamp 36 Brie wate O Loopamp UPILAT ASS AERS S Loopamp ITY RRT Y PARNE BB LAT YF GEBREM O5CMA Ry KRY RY MY O E FIOy7 CRAB LER O SOMRIRSPRE CORR 240 260nm 350 370nm 5 O Als ORR NSH BH x5 MBS RAB LERE FRIMRERSIORAICDUVTId Eiken GENOME SITE CURL http loopamp eiken co jp amp CBR lt ESA 2 Primer D2 LAMP SAIC KSIBHBIC E o CME primer RH OSSGAFCROKITOC Be ICAI oTi LAMP SABA primer KERIB I KACHMA lt IES primer s ZEY Z Hd Net Laboratory LAMP SA DS X B IR D KIIP Primer Explorer http venus netlaboratory com partner lamp CBR lt IES HE primer SROTFLU KICDUITIA LAMP 3AOIBS primer OHHREDSU ECRMURBIR lt EO RMBACRELES HOC primer D RAZDY TY JeBHC UCEKS SRSISMBAS ABRIL FILE CHNMSRST DAE CTD primer AREF SRRURERIA DILES FIP BIP ICDUVTIA HPLC 88 TU FKICKSSMEHRLET 3 MRO 1 20C TRF LUT
3. 4 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 4 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 Contami nation 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 1 LAMP reaction is very sensitive and even the slightest amount of amplified product tainted into the reaction might cause false result Therefore 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 wit
4. No 1 150 154 2001 4 Tomita N et a Abstract for The 73rd Annual Meeting of the Japanese 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 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 Manufacturer EIKEN CHEMICAL CO LTD Sike 143 Nogi Nogi machi Shimotsuga gun Tochigi Japan 3LP2419 D CORBEEL lt iA CO SIPRUT lt KIERU FR 2008 F1 BAZI CB 4 hh Wats 2006 F4AMa 3 hk 4 Loopamp LAMP Loop mediated Isothermal Amplification 3 RNA te 0g a8 Fy h C RT LAMP CHa LAMP Loop mediated Isothermal Amplification i4 0 1 BHOBROKE E3 JUTR FIM ROOSR CETIS 6 mAAR S 4 EAO primer amp E3 D BS SEORREMS O IBBAROS lt ASEC SBE DS lt BABRWICBLTWIS D0 SORBAS I SHU VABFISIBATS A y hid LAMP SA AAUYC RNA fei CRBS ISIE ST S RT LAMP RMIODIED GAde ty bh CS Hea SHARC DNA BARERA ALIZ Enzyme Mix 2AliS CICK DYPILCHRe ERE
5. U CMUBLTCIERU f LAMP method is a novel isothermal nucleic acid amplification method ota 0 uL tes 2 REOMRABWSCSH BBC IDK ECHOTCIERU BRE eg using 4 kinds of primers and DNA polymerase with strand displacement R M Ui ee eas Pe ene ee S es ERER nena activity Among 4 kinds of primers two of them are inner primers whose 3 x For visual fluorescence detection also add 1 u L of the Loopamp rales PRAPA IAUC I A sea aceite ONORE RECU DRA TEDA ADR aD F end and 5 end are respectively designed to be complementary to two Florescent Detection Reagent available for sale separately and maintain E TARE UBEAZCUIIYUTMSCRA lt ESU BB Enzyme Mix woe se BS Hi Sark swears RRI E different regions of the target sequence When the primer linked strand the total mixture amount at 20 u L EM AEST SENDHOSTOT BML lt HIELUITCIESU re i i i z CEM IRSD BODES WU lt HULLT IES 485K UMP2AA is replicated resulting the 3 end structure to self anneals to the comple se When used in combination with Loopamp Primer Sets follow the 2 WtBRBLORSOZRA Loopamp RNA itat wy F mentary region in self structure it forms the loop structure at the end pens i 3 5 ees rae x z Ames i k RT anes 75 723M 206 14 LMP245 This stem loop structure will allow the 3 end to initiate self elongation preparation instructions of each primer set to prepare the master mix DUTIVE BORIC A a SOte ccs aad 192 F2 LMP246 and another inner primer to anneal to its loop regio
6. Y KO LERHERUCTRASTEDHO RNase ICAO GAICDBENTLEWET RNase SMA CH SMA CMRI i 1 The following amount of the component is required for one reaction ETM SOBAAFA TERIMORNRED SH LIED F1 JOREE RE OAAS RRR MK k SSCARRSASOMMOTNSEBA Contents of the kit 48 tests 96 tests 192 tests EETAS BASREUT BMIY KO LERMIY KO LO MAD ROT SKSIT FSONMBOET RNase FRE RILI RE TERRIA SLU 1 2x Reaction Mix RM 0 6mL tube 2tubes 4 tubes Reagents gt Smuts TEULTCEAU th eS ee RNase DIBA SB CMEBDHO WEKOERBDWMETS 2 Enzyme Mix 3 EM 50uL ltube 2tubes 4 tubes 2 x Reaction Mix RM 12 5 wl 4 Y FAN I ld Loopamp SENESE UPILITA LY RMR YA D RNA RRZT SRS ORRER SHCRATS 3 Distilled Water DW 1 0mL tube 2tubes 2 tubes Primer FIP 40 pmo ED TROT Y IN S CREED 0 5 C IMA hy KRY RY RD RRICRATSFI TSld MESNE COCOSRAT D 4 Primer Mix RNA 4 PM RNA 30uL ltube BP 40 pmo BRUSCCMCaET RERICHAT Skid DEPC MIB2kKEC RNase 7 OKEAUS 5 Positive Control RNA 4 PC RNA 60uL 1 tube z ee S pmo S63 BIG BRR tS AA VEBE TSAS HAEC EAA Loopamp RBE FR VAITE L RREBSOBRKOFDSO ei pmo sk ft os 2 5 3 A pmo St BRM ARO Se CEIR lt IERU RNase DORA GBS x1 The notation on each reagent tube is shown in B3 5 pmo 4 BS CoRY 5 Loopamp RiiFa D VAS SyIABRAMAF a FDICISUV RH LBL 2 composition Qx Enzyme Mix EM 1 0 uL MBE MICKSIYISR Y BVIC DEBOL RETTES TES UVRHICKSEE BBS CROKBRESESTRBADHVET era pH8 8 5 ae Distilled Wat
7. reproducibility of the amplification can be BSED Loopamp Hi SMRWMRABISCC CHRNEDTAETS RER ASS AIDOSYPERBAROMEES TEDE TEDE EO cation efficiency and enables amplification within a shorter time It obtained The first screening for appropriate LAMP primers might not PIBRMORTA KOMEN E RE 240 260nm 350 370nm A SHA ICWETERARC LET OC ROMGSBHBICANSCCABIITC lt ESV produces tremendous amount of amplified products which makes simple necessarily require highly purified primers However after the primers RDF IRBA N SOMBER LC RMF 1 FORDE BRBS CRE FERIPDNSY PERSWENHSCA UFHSAREBIO DBOR silat Saari PRD eee y EEA arg deter minsa fiance to use purified FIP and BIP through pe to r r puritl fon LIKE TAR LET BEDY KO LEMRICREORNERT NSB NILE EDIT CWE LTC ESV be UDI to 3e Se eae roe eee eee iy target PINA Byte A A A EE o wis Rae z B oor LAMP method RT LAMP By employing the Enzyme Mix containing reverse 3 Reagents preparation IRM OY KO LER RICHER LAINE LET 3 ERDD SRE IES CORA BURIC IN TARE T V Fee aN lie 2 otra anI DNA ae a Es Gy ET ee en WED 320nm HAORS BMCEBMEHUCBZSCENBNDETOT FREE TSE cation can be accomplished at a constant temperature 60 65 usually temperature Once the reagents are thawed keep them on ice BIY KOL SHED Y KO DELERRU THIS UTS IER IE ROME 4 RNA DFIAIERICAAEBEOD MIRUITIAERBDMETS IC RNA aaa 63 C ina short time 1 hr for standard in one step 2 Preparation of master mix Operate on ice RN RBOW NDA SSID
8. room temperature and keep them on ice for 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 Enzyme Mix EM 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 1 Only use the specified Loopamp Reaction Tube for turbidity or lorescence detection Other reaction tubes might have different optical ransparency and can cause misjudgment 2 Take full care when handling reaction tubes as they are vulnerable to scratches or damages 3 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 malfunctioning
9. 3 Detection In order to prevent contamination the following procedure 1 or 2 is Cc 1 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 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 angle of th
10. 60 65C WF 63 C Ea GREET 163 O1 AFYFTCRT LAMP RMATSCEMTCART Fy FAA A87AND Q96TAMD 1927A ND 1 2x Reaction Mix RM 0 6mL Iltube 2tubes 4 tubes 2 Enzyme Mix EM 50uL 1tube 2tubes 4 tubes 3 Distilled Water DW 1 0mL 1tube 2tubes 2 tubes 4 Primer Mix RNA PM RNA 30uL 1 tube 5 Positive Control RNA PC RNA 60uL ltube X1 C Ald RRF DICeHAN THISRMTCI x2 ABR 2x Tris HCI pH8 8 40 mM KCI 20 mM MgSO 16 mM CNH4 2S04 20 mM Tween20 0 2 Betaine 1 6 M dNTPs 2 8 mM each X3 Bst DNA Polymerase amp AMV reverse transcriptase v 7 AZLIEBOTS X4 96FAKDC 192 FARDIClad Primer Mix RNA RU Positive Control RNA SSENTHOECA CAERE LAMP id 4 280 primer CHS HSM AID DNA Polymerase FAUV CRIME FORRES RRS ACI 4 AMO primer D55 2 RO inner primer dh ED 3 AE 5 BU CHAE PORCS 2 IAs TS primer T 5S MOBIL ED 3 AP SOPRRM CSM UC tE HE BIA IC PILI SKIARELET c c TSB RMA CO inner primer CKOERST SAT AIL TPRiED SORBCIPRRM gt IL PRBAICMIEICP IL UIE inner primer DOOREREKRMARORT COETLEST THICKY LAMP SAISAUISHRD 1 BH CHSICEMBNSS D0 Fme ERRUA KE RNA AREE LIEGE OSD UMGRSBRE ZDT cDNA DEKD SRBOBIBET RT LAMP RD 1 AFYIT TFICCMCEST BB Ri RIZOEMICDUVTlIa Eiken GENOME SITE CURL http loopamp eiken co jp ACBIR lt EA CFR A
11. I TPaERLIERA EBEDET 4 a ROA me 730 as x sacs DRR oe S secondx3 times The prepared master mix should be used as soon as E Ge ae 5 A ZS th 230 GADFEMERERIOTSA EEEE 2000 Haw touse s ssible 6 SB RA 5260 DFEMF RFAJOJ IDR amp 2003 i i 2 RIF A TFISTRIBLOT WOT HURUVICIBEBUT lt ESV i i is Sones Ai T i 5 an ROE 1 Materials required but not provided F 4 Operation procedure ae L Lex att s Tepe agamine A etal Wolecularand Vellular Probes 20 NO 3 Sterilized tubes for master mix preparation 0 5 mL 1 5 mL Pe 3 RMFA TPMSASAMICHA EC SDRVC CARR CHBLTCIERU R ge 8 BAMBLA 1 421 IF 1 ZRA GAMENET 54 No 3 667 715 1999 icropipettes 0 5 10 uL 10 100 4 L 100 1 000 u L 1 Mixing t aster miz and sample solution Onerate on 1ce Fi TEZ CELSRHSCIEUK lt AE CSRUELDOD FA POBIAIC KOREASRISTRMMHOLST Loopamp BENERE UPIVSTA TY EMT Yb ORMID y DAT A DORR LIES IA RUDY AA A at L BRAKES A OMIEORACROET RHMCFRAAL PSMA 4 RMFA DICVAI SVI2A GUPIVBERONARMENTISTCA eF DU A4 PIL fG 0120 308 421 TFEOMBLR CHER LTCIERU Pipette tips with filter 1 Dispense 20 uL of the master mix into each Loopamp Reaction Tube O oO available for sale separately K K AUSGDCH 2 Loopamp Reaction Tube vai r sale separately O O O Aluminum rack for cooling tubes 2 Add 5uL of sample RNA to the master mix and the volume of the solution Ice
12. U BRR AEn CHRUL APRRIABSICKECRELET 2 VAS SyvIAZORR CKRETHDT lt SIESU 1 BIRRA LEV AS S y DARRAREF 1 TICSMREWBRT ALBA BROEIS 1 FAZARBED CHIELES O FUTILE I lt at gt lt Ais gt 2 x Reaction Mix RM 12 5 wl Primer FIP 40 pmo BIP 40 pmo Loop F 6 20 pmo Loop B 6 20 pmo F3 5 pmo B3 5 pmo Enzyme Mix EM 1 0 uL Distilled Water DW X ul GBs 6 at 20 0 whL FAb x6 WSF ULSBMBHO EAD Loop primer ANS cc CiBlBRAAW 1 3 Ic MSN O DY hO LR lt at gt lt Ais gt 2 x Reaction Mix RM 12 5 uL Primer Mix RNA PM RNA 2 5 ul Enzyme Mix EM 1 0 uL Distilled Water DW 4 0 uL 6 at 20 0 uL FAb SLB RRWORSIA LieVRZI SyIAICBFO Loopamp Bt BRE MRE luL RMU PII 20uL ELUTEEL x amp D51V Ry hcMSNe CHEAT SRS VAZS SyIZOBRISSIS1 V By FORRICHD TCE 2 DER FA PeR lt ROMUCRSTS MR Pye VIEWS A NISSHEBA BSVVMANLFyIAZSE ICT 1 WA x3 BORMRICKO D ROLE MBB ABRMWBICRMOIT UR ZEYVSODYCHS CN ZSI ZVYIRZEVET MILF Y IAZSFO COMPIDGRICTISC BRD KET SUREMHOVEITOC 1 WAX3 GaRPUT lt KESA BH PR LIEVAZS SyIAldT SICEBUT lt KIESU 4 RIEA 1 VZS SvIRZCVYINBERORS CKETH2D TIES 1 Loopamp RMFA Dle CYALRMABCDY JUROR SROVAY SyI7A2uL DiELET 2 DY TILIA 5 ul BAIL 25ulL cl
13. as KEDYE IRIDA UT a BIY FO JLICld Distilled Water DW 5ul BIY KOLITI Positive Control RNA PC RNA 5uL i AlLAD COCR ENyF1 VD Mid e y PARDEE TOS y CYTICKOR lt BSELER ZEYVYIDYLE F KE RAORARVAMWERWKADISEBLES 2 i8080 1 Loopamp BENERE PISTA TY RNT Yb XT YF BEREND 05 CUA hy KhY RYE ICR DERAORM Fa Deeyby 60 65C 63 C T 30 60 DAT YFAN FU HS CREt LIE primer CKD CRHMBRSOC BSD UCORHRAID WET F 80 C 5 DARA 950C 2 DMT YFAN FTSCCICKYO BReK BA RMARLACES 2 172 Read this instruction carefully before use 3 RH 2 RIMEOFA Dia v vy JERITE HAMMERS TEDER
14. ation E7 GB MBC Cla Eiken GENOME SITE CURL http loopamp 1 LAMP RiiIGIERICHRA RICO BEENS DNA DC lt METEIBA RNA Am p lification Kit unction and condition for UV irradiation refer to Eiken GENOME SITE eiken co jp ECBIR lt IERU EE HA ORMIGE OMMHBBE CSIR FECRILBRESE ST RACRSENDGVST COKIBIVISR CURL http loopamp eiken co jp e lt IE amp U YaYEGRISKO MERUIY TLOBMSTRGIRO DU YAY FEE CRT LAMP 2 Primer design D LYEKTY BERE SAL MUI CRSEOMBRORUWM TOD BER UPILSTA TYR Characteristics Appropriate primer design is essential for amplification by LAMP Use Loopamp LY RRT Y MSSRERBSRSCe LY RK YEKCOSE RIY WIPES RBH CGI TIES LAMP Loop mediated Isothermal Amplification method is a novel gene exclusive primer designing software for the primer design Refer to LAMP BENTES AMIR BOMBRMBLE CHICA Sf SERNSRMF 1 TORS CIMIBEMERMATROIRSIA BR amplification method capturing the following characteristics Only one primer designing software PrimerExplorer at the website http aS Oe aa Hy SABRE RIDE CS E enzyme is required and the amplification reaction proceeds under isothermal primerexplorer jp e BB LY RRI Y hOS SS CMAN ORTER KA RO IA lB On CSE EH condition 2 It has extremely high specificity because of the use of Using the highly purified primers a rapid gene amplification can be k 3 SHERRY 2 SREB CRRA RE SAT SRS DY TK OMNASNS RMR 4 primers recognizing 6 distinct regions on the target It has high amplifi performed and stable
15. crushed ice and ice box should be 25yL in total For control reactions use 5uL of Distilled Water Centrifuge for micro tubes DW for negative control and 5uL of Positive Control RNA PC RNA for Centrifuge for 8 connected tubes positive control Mix the solution well by pipetting or tapping the tube with O Vortex mixer the cap closed and then spin down Be careful not to cause air bubbles 4 ISI RMICRUCOBRra ixi a ey VIIA WIC Ra SEIE lt For real time turbidity detection gt when mixing ZA SVYIAZCTYIIVERARWEA RINE VAD CHSCHEAEO Loopamp Realtime Turbidirieter 2 Amplification reaction FCROBYEORBNCROEIO WIMtEVAWKDERBUTCKIESU SE EF as Baia TAA C1 Set the reaction tubes in Loopamp Turbidimeter Realtime or End DR TOSBAA AEVIDYUUTRMASMOPBUVT CIES or end poine turbidity detection z Point or the incubator with hot bonnet temperature accuracy within O Loopamp End Point Turbidimeter 05 k 8 i 5 SACORMF 2a TPORE 0 50 and incubate them at 60 65 C for 30 60 minutes The 1 RMBOF I PeREPSRMOWS ESCRI INF r y IMD EAKS lt For visual fluorescence detection gt ieee Renee is Spenn eae the ciare erue a the primer BICROWLTCCKIERU RROMI YS IR a VIABMIEO k X O Loopamp Fluorescent Detection Reagent i me use it erefore examine the optimum condition be ore an E SOTAA RERE Cee eee RI es y DUG FREE E Loopamp Realtime Turbidimeter Loopa
16. e 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 3 VS VY 4 aution for use Handling the kit 1 This reagent kit should be stored at 20 C To prevent the reagents from 2 3 VU 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 Thaw the reagents at
17. er DW X ul adq s RWA 0 5 2 0 uL ZAVT 2 BEOPHAO ACHARMAAWET 6 Ey hid PHAR BWOHIC CHAK IER MgSOq 16 mM Total 20 0 uL test TFVOLTIOV K EtBN AZt SYBR Green I TELET 7 BRFRADNMCRRECERIGG BABROUHEARSERHDHVET NH4 2504 20 mM ak i i Kain ih bat mice eee a Cian te aN 2 i to aaa A on ee ea ote o 6 Loop primers are not necessarily required However the use of Loop SIBANIEBAIS LAMP ROTERES AOR IR LO SRSIEN DT APY KORACH o THER F RAO PRE LICR MTS OBSO iil re e primers shortens the amplification time by about 1 3 BREOMA CHECEDS BAK NS VSSI NS VERES RCRA SRIBLT CIES FN een 2 ps 8 EEY KOMBEICHR LAB GFARE RBOSE CLIR TER i Control reaction Ri LOBaSR HE KECOWECHRUCHELESHICMULT SHlt WOBREEAWNE x3 It is a mixture of Bst DNA Polymerase and AMV reverse transcriptase lt Reagents gt lt Amount gt 1 BRORRU th 2 x Reaction Mix RM 12 5 uL w yF 20CT E h 4 Primer Mix RNA and Positive Control RNA are not included in the 96 Pri Mix RNA PM RNA 2 5 uL D KAY MAWF 2OCTRELTCESU MEORICSBILTSEOIC EES SMBICERORABREMR Exp Date PICAILT lt EEN ani A ane ee au y stet gt uw utaha Bni oym pa as A a SIR EBURIC GFE SOUL CCRRICIECU CREEPS 20 COE 410 HRFI JAPP FyhT AGMEECMECU TVET ROMER Principle Distilled Water DW 4 0 uL Zit t ae Me ED AAN SY F E Sthg o TEER TIA MROA CSECACROSNCHVECAM BAO RAIS SEMPSICEIS SERV IKDSBMIDRSOSBIBAIICHL SHBOREICH rinciple a e eee HHSDIEDIGRID T lt ESI
18. h 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 2 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 3 When handling the sample always abide by the biohazard counter measures 4 Since RNA is vulnerable to RNase handle with care There is possible contamination of RNase from such as sample specimens blood and urine etc tissues experimental tools reagents water the operator s saliva or perspiration RNase is stable against heat and might maintain its activity even after autoclave sterilization In order to avoid RNase contamination the following points should be concerned Designate exclusive operation area and tool for RNA treatment Use sterilized disposable tubes and pipette tips Use RNase free water for example DEPC treated water The operator must wear gloves and masks to prevent RNase contamination to the specimen from his or her saliva and perspiration 5 Do not expose the Loopamp Reactio
19. mp End Point Turbidimeter or 2 Inactivate the enzyme and terminate the reaction by incubating the RACESISD CE RR COCDESRL SRERSUAUIRO Mi xk BIG AR ITIL T Zr a incubator with hot bonnet temperature accuracy withint 0 5 C mixture for 5 minutes at 80 C or 2 minutes at 95 C OR CIE UM HERDS SNE K BDIET BASBKASBSAMDSA143 Sih 2 2 1 2 3LP2419 D
20. n Tube master mix preparation tubes to UV light A change in color or degeneration caused by ultraviolet lamp sometimes results in misjudgment 6 This kit is designed for research use only 7 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 8 Eiken Chemical Co Ltd does not bear any responsibility for false judgment or any consequential damage derived from the false judgmen caused by non capability problems such as operation error 9 Use the kit before the expiration date which is labeled on the outer box CExp Date 10 The reagent tube is made of polypropylene and the main material for ki 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 Product Name Unit Storage Expiration Code No 48tests LMP244 x near a A Kit o6tests 20C 1year LMP245 192tests LMP246 References 1 Notomi T et al Nucleic Acids Research 28 No 12 e63 2000 2 Nagamine K et al Clin Chem 47 No 9 1742 1743 2001 3 Mori Y et al Biochem Biophys Res Commun 289
21. n to synthesize new DNA 2 After dispensing gently tap the tubes for a few times hereinafter CTE buffer SAUVAUVC lt ESL REFU MESSMO RMRICASCV YAY strand with strand displacement manner Through the repetition of these rae tred tae ts 7 inad ormie solution byrepeatedly nversiha the LYRE L SN BHOBRICHME lt KRHARLET KEDYAIIC Seva processes the amplification proceeds and this enables amplification to tub Roh Ae eo Cabot one ti y Aft gt Ca Zn Fe AY SORBET AY AS SIC S0RSECRHEORAICEOVETOT lt continue under isothermal condition with only one kind of enzyme When HE OE mes Y VONE TAERA T aout Secon ESTANSE MIXING BELTCEAU 1 Notomi T et al Nucleic Acids Research 28 No 12 e63 2000 RNA is used as template by initially adding the reverse transcriptase one well centrifuge the tubes for a few seconds hereinafter referred to as r 2 Nagamine K et al Clin Chem 47 No 9 1742 1743 2001 step process from cDNA generation to DNA amplification can be achieved spin down And the mixture can be used as the master mix for the 3 RAF 7NR A 3 Mori Y et al Biochem Biophys Res Commun 289 No 1 150 154 2001 For further details of the LAMP method refer to Eiken GENOME SITE reaction Notice that too much mixing by the vortex mixer might 1 SRRY BB RMRLOWSd RMF a PSMSSAO Loopamp RAF as tiie Fi i CURL http loopamp eiken co jp e inactivate the enzyme and assure that vortexing is conducted at 1 JETAS HEWORMF
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