1、9K +4nGrP5b h*(=24 7300/7500 rnG PCR 1T 4nG/_rP; E)AAu4nGrPE 4nGhSd4nGDBPrimer Extended on mRNA5 3ReversePrimer5Synthesis of 1st cDNA strandcDNAOligo d(T) or random hexamer3 5cDNASAMPLE DOCUMENT 3 30, 2004 9:28 , GSG_FrontCover.fmii 7300/7500 rnG PCR 4nGrP9K + Copyright 2004, Applied Biosystems. All
2、 rights reserved.For Research Use Only. Not for use in diagnostic procedures.Authorized Thermal CyclerThis instrument, Serial No_, is an Authorized Thermal Cycler. Its purchase price includes the up-front fee component of a license under United States Patent Nos. 4,683,195, 4,683,202 and 4,965,188,
3、owned by Roche Molecular Systems, Inc., and under corresponding claims in patents outside the United States, owned by F. Hoffmann-La Roche Ltd, covering the Polymerase Chain Reaction (“PCR“) process to practice the PCR process for internal research and development using this instrument. The running
4、royalty component of that license may be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents. This instrument is also an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limite
5、d PCR license in accordance with the label rights accompanying such reagents. Purchase of this product does not itself convey to the purchaser a complete license or right to perform the PCR process. Further information on purchasing licenses to practice the PCR process may be obtained by contacting
6、the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404.DISCLAIMER OF LICENSE: No rights for any application, including any in vitro diagnostic application, are conveyed expressly, by implication or by estoppel under any patent or patent applications
7、claiming homogeneous or real-time detection methods, including patents covering such methods used in conjunction with the PCR process or other amplification processes. The 5 nuclease detection assay and certain other homogeneous or real-time amplification and detection methods are covered by United
8、States Patent Nos. 5,210,015, 5,487,972, 5,804,375 and 5,994,056, owned by Roche Molecular Systems, Inc.; by corresponding patents and patent applications outside the United States, owned by F. Hoffmann-La Roche Ltd; and by United States Patent Nos. 5,538,848 and 6,030,787, and corresponding patents
9、 and patent applications outside the United States, owned by Applera Corporation. Purchase of this instrument conveys no license or right under the foregoing patents. Use of these and other patented processes in conjunction with the PCR process requires a license. For information on obtaining licens
10、es, contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, or The Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California, 94501, USA.TrademarksApplied Biosystems, MicroAmp, Primer Express, ROX, and VIC
11、 are registered trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.AB (Design), ABI PRISM, Applera, Assays-by-Design, Assays-on-Demand, Celera Genomics, FAM, iScience, iScience (Design), and MultiScribe are trademarks of Applera Corporation or its subsidia
12、ries in the US and/or certain other countries.AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.SYBR Green is a registered trademark of Molecular Probes, Inc.Microsoft and Windows are registered trademarks of Microsoft Corporation.All other trademarks are
13、the sole property of their respective owners.Part Number 4347963 Rev. A3/2004,)7300/7500 rnG PCR 4nGrP9K +v4nGrP0#/iii!?viiV)S* + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiV)9 C . . . . . . . . . . . . . . . . . . . . . . . . . . .
14、 . . . . . . . . . . . . . . . . . . . . . . . . . viiiV)9 u . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii|,X? A E4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii1 1 01T 4nG/_rP1VE . . . . . . . .
15、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Gb 7300/7500 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2Gb4nGd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16、. . . . . . . . . . . . . . . . . . . . . .2Gb4nGrP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34nG/_rP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61 2 0AAu4nGrP110#/ . . .
17、 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11VE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12An4nGrP . . . . . . . . . . . . . . . . . . . . . .
18、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12E:9; . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13E!9“!9“ E)-6. GJJ h (RT-PCR) . . . . . . . . . . . . . . . . . . . . . . . . .14EvJ (= . . . . . . . . . . . . . . .
19、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151 3 0; E)170#/ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 RNA + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20、. . . . . . . . . . . . . . . . .18 RNA E cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .191 4 0E 4nG hS210#/ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21! . . . . . . .
21、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 PCR =rNX#A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22vi 7300/7500 rnG PCR 4nGrP9K + hS . . . . . . . . . . . . . . . . . . . . . . . . .
22、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .234nG (AQ) hS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24n 4nGrPd,X?U-0J , rPx ,54mn +n|K5xs MicrosoftWindowsXP 024xK) DNA RNA PCR ,X8-A T6xKG.,PE| ) *b|9 )K9 )q E,X Q_VEFile Open Spot Set,X
23、PCR #!oG*rd“,X GLcnG# G*4 /#d +E4nG (AQ) rPdGJWrP#d,XAA K5b h*(=24 c#24: +(SDS Chemistry Guide) 5b h*(=24 7300/7500 rnGPCR6( (Online Help)Gb4nGdn4nGd*b.Bn- , GLc,X4Gr PCR d4nG (AQ) dr PCR ; r PCR dE/PCR =r, |,X, PCR E KE DBGL5 PCR 434 =rJ?r PCR 4p8rPB| EE-0J=r B9.BnrPB,X s S*4nG hS9,|L/Ld 1! HH (AD)
24、 d,XrDB*bL/Ld 1! HHd,X4nG hSL?U4Gb4nGrP4nGrP0#/7300/7500 rnG PCR 4nGrP9K +3“G1Gb4nGrP4nGrP0#/4nG (AQ) rPA4nGrPd,XHE/ 2 GL (RNA) *f97! 2 GL (cDNA) E)CK“|)B5NO,Xy !89;t TaqMan vJ,X 5c0W ,X9; 0(M=r,X/(=!7 y 9;z(Rn)L !9; z,!EW,Xy 9; zDelta Rn(Rn)4n,X4 PCR 5* ,XGRn= Rn 4 -G,X *bX4- |?U#n,XS,X S,XG-0 5 10
25、 15 20 25 30 35 40GR0757Gb4nGrP* Lo,X$m7300/7500 rnG PCR 4nGrP9K +5“G1* Lo,X$m*G?US* MicroAmp : h1u ABI PRISM: h1u S* ABI PRISM: h,S?Z,J,y,X+ U n S*,/ ! d4nGrP8Vo/_rP/_rP )4nG hS*DB hS 4 S*Lc 7300/7500 PCR o,XECDBE d4nG/_rP4nG/_rPE/7300/7500 rnG PCR 4nGrP9K +7“G14nG/_rPE/1.AAurP K1 2 0a.n- 4J.BnGDb.
26、AvC b TaqManvJ:9;,XAc.S* Primer Express(=AAuECAAu(= vJ2. RNA K1 3 03.S* High Capacity cDNA Archive KitG cDNA gA, RNA * cDNAK1 3 0a. 7 +b. hS,X! h(/,X# cDNA g hSx 50 PL E) (RT) NX#Ax 30 PL GLGJ“x 20 PL RNA .Bb! 50 PL PCR =r h RNA E cDNA ,XBG 10 7100 ng/ 5 PLRT Master MixRT NX#APL / 1 hPL / 7 haa.! RT
27、 h/ 100 PL K!9Px3bb 104 PCR =rh K!9Px4,X! hVpL?U 5 PL cDNAL?U 6 E) (RT) hh Nq,X/C*b Nq E) (RT) h9/#E/E,X3JLNq,X cDNA *bg10 Reverse Transcription Buffer E)4#10 7025 dNTP7! 8.GL42810Lc(=10 70MultiScribeReverse Transcriptase E)GJ50 U/PL535 GLGJ“21 147Au50 350GR23221 1 01T 4nG/_rP4nG/_rP8 7300/7500 rnG
28、PCR 4nGrP9K +“G.c.S*!9“ E)-6. GJJ h(RT-PCR) “4,X NM DAAu 4nG (AQ) rPd z ,| 4nG (AQ) ,nG(RQ) hS,XC f,x6G?UVp|,XrPS* hS,Xh(!8!9PxA?U+9)?US*,Xh(/c | +9S*,X h(G+9S* h(,XA4CA K6(1 1 01T 4nG/_rP4nG/_rP10 7300/7500 rnG PCR 4nGrP9K +“G9.d4nGDBA K1 5 0a. )EAnalysisdAnalysis SettingsdA5BA5Bd DS* Auto Ct7| CtE
29、NMb. )OK da dDBc.Vp?U|AH 4 Kd. )OK da dDBe.,d4p4A B4E |#4 1 ,X RNase P 2 GLGJ P ,XGD 50004 2 10000“GGAA K1 32 NIG!5BdA5B“GGAA K1 33 NIAH4 K7300/7500 rnG PCR 4nGrP9K +11“G2120AAu4nGrP0#/Primer Extended on mRNA5 3ReversePrimer 5Synthesis of 1st cDNA strandcDNAOligo d(T) or random hexamer3 5cDNAK1 12 N
30、IK1 12 NIK1 13 NIK1 15 NIK1 14 NI1T 4nG/_rPVEAn4nGrPE:9;E!9“!9“ E)-6. GJJ h (RT-PCR); E)E 4nGhSd4nGDBEvJ (=AAu4nGrP1 2 0AAu4nGrPVE12 7300/7500 rnG PCR 4nGrP9K +“GVEL _,X4nGrPAAuG*4 )PCR=r! h )(=t TaqMan vJ SYBR 4 9;FEAAu4nGrPL5 5c GLGJ#dSYBRGreen I AAS* SYBR Green I 9;/ J DNA 4 9; PCR )K PCR (=6.L,X
31、 PCR (=E # d E) (RT) PCR =r|?US*,XAG!5B b|E,X!9“!9“ RT-PCRx!9“ RT-PCR ,X h; Oj RNA E) cDNAEEPCR cDNA E =rE/“E b# ) cDNA S *,XE),1 cDNA 9S* S*AmpEraseUNG GJ9L!6!_+-“5x !9“ RT-PCR E) (RT) PCR =r 4#24 * L1T ) S*A1u G .Q RT PCR =r 5 !9“ RT-PCR |6S*!_+-e GJ AmpEraseUNGuracil-N-glycosylase G J-N-2 GJGUNGG
32、J,XA4AA KSDS :+!9“ RT-PCRG?U +n4nGrPAAu!9“ RT-PCRGLtENM,XAA KSDS: +!9“ RT-PCRGR2333EvJ (=7300/7500 rnG PCR 4nGrP9K +15“G2EvJ (=,cE+vJ (=5b h*(=24 EvJ (=o/Ex Assays-on-DemandGene Expression ProductsAssays-on-Demand E)0#/Primer Extended on mRNA5 3ReversePrimer5Synthesis of 1st cDNA strandcDNAOligo d(T
33、) or random hexamer3 5cDNAK1 18 NIK1 18 NIK1 19 NI1T 4nG/_rP RNAAH RNA #z; E)d4nGDB RNA E cDNAAAu4nGrPE 4nGhSPrimer Extended on mRNA5 3ReversePrimer5Synthesis of 1st cDNA strandcDNAOligo d(T) or random hexamer3 5cDNA1 3 0; E) RNA +18 7300/7500 rnG PCR 4nGrP9K +“G RNA + RNA5b h*(=24 o/ s$m RNA ,X #44
34、 O46(=$m RNARNA,XBG.B| 4nGrPS*,X RNA 0 5xJ A260/280!)Pb 1.9x*6+ “?H3,Xx E) (RT) PCR e High Capacity cDNA Archive Kit ProtocolG cDNA gA,rP(4312169) RNA S,XLtAAH RNA,X#zHigh Capacity cDNA Archive KitG cDNA gA,rP4E 0.1 7 10 Pg,X RNA E cDNAb! 50 PL PCR hhCG,X RNA E cDNA cDNA ,X4#z 10 7 100 ng / 5 PL24B
35、ABI PRISM6100 Nucleic Acid PrepStationABI PRISM6100 GL 6100-01 RNA :ANucleic Acid Purification Elution Solution GL “7$#Nucleic Acid Purification Lysis Solution GL ?$#Nucleic Acid Purification Wash Solution I GL “# INucleic Acid Purification Wash Solution II GL “# IIAbsoluteRNA Wash Solution (DNase t
36、reatment)AbsoluteRNA “#DNA GJ)43058934305895430589143058904305545TempusBlood RNA TubesTempus# RNA A1u*bS* 6100 PrepStation dGL0n RNA # RNA A1u G5$rP4345218 44 RNAIsolation of Total RNA from Plant and Animal Tissue Protocol(= |(=44 RNA rP4330252Primer Extended on mRNA5 3ReversePrimer5Synthesis of 1st cDNA strandcDNAOligo d(T) or random hexamer3 5cDNA RNA E cDNAS* High Capacity cDNA Archive Kit G cDNA gA,7300/7500 rnG PCR 4nGrP9K +19“G3 RNA E cDNAS* HighCapacity cDNAArchive KitG cDNAgA,S* High Capacity cDNA Archive KitG cDNA gA,B 4322171!9“ RT-PCR ; 1!9 (RT);High Capacity cDNA A
