PrimeScript® RT-PCR Kit

1 RR014A. PrimeScript RT-PCR Kit . v201110Da PCR Polymerase Chain Reaction DNA 2 . DNA PCR DNA RNA . RNA cDNA PCR RT-PCR . RNA PCR RT-PCR RNA. cDNA RNA .. PrimeScript RT-PCR Kit 2 step RT-PCR . M-MLV RTase . PrimeScript RTase PCR Hot Start PCR TaKaRa Ex Taq HS . RT-PCR .. RT-PCR . 42 RNA . RNA .. RNA cDNA PCR cDNA .. I. 50 1. 1. PrimeScript RTase for 2step 25 l 2. 5 PrimeScript Buffer 200 l 3. RNase Inhibitor 40 U/ l 25 l 4. dNTP Mixture 10 mM each 150 l 5. Oligo dT Primer M 50 l 6. Random 6 mers 20 M 50 l 7. TaKaRa Ex Taq HS 5 U/ l 25 l 8.

2 10 PCR Buffer II 250 l 9. Control F-1 Primer 2 20 M 10 l 10. Control R-1 Primer 3 20 M 10 l 11. Positive Control RNA 2 105 copies/ l 20 l 12. RNase Free dH2O 1 ml 1 20 l PCR 50 l 50 . 2 Positive Control RNA . 3 Positive Control RNA .. Random 6 mers pd (N)6. Oligo dT Primer dT 4. Control F-1 Primer 5'-CTGCTCGCTTCGCTACTTGGA-3'. Control R-1 Primer 5'-CGGCACCTGTCCTACGAGTTG-3'. 4 TaKaRa RNA PCR Kit AMV Ver. RR019A/B Oligo dT Adaptor Primer M13 Primer M4 . 2 RR014A. Positive Control RNA . Positive Control RNA SP6 promoter pBR322.

3 Kb pSPTet3. SP6 RNA Polymerase in vitro transcription .. SP6 Promoter Tetracycline Resistance Gene Hind III BamH I Sal I. EcoR V Sph I. F-1 Primer R-1 Primer 462 bp 1 RNA Control Primer .. 1 authorized instruments . TaKaRa PCR Thermal Cycler Dice Gradient TP600 . 2 . Agarose L03 TAKARA 5003/5003B . NuSieve 3:1 Agarose 50090/50091/50094 . 3 . Mupid -2plus M-2P . Mupid -exU EXU-1 . 4 . 5 . II . 20 . 3 RR014A. III . 5' 3'. 5'. mR NA 3'. Synthesis of first strand cDNA. with PrimeScript RTase 3' 5'. cDNA. PCR components added.

4 Second strand cDNA Synthesized with HS. 5' 3'. 3' 5'. Amplify cDNA. 5' 3'. 3' 5'. 5' 3'. 3' 5'. 5' 3'. 3' 5'. 2 PrimeScript RT-PCR Kit .. dNTP mixture RNA .. 65 5 min. 4 .. 30 10 min.. 42 50 15 30 min. 95 5 min. 4 . Random 6 mers .. TaKaRa Ex Taq HS PCR . 94 30 sec. 55 65 30 sec. 30 cycles 72 1 min. / kb 3 PrimeScript RT-PCR Kit . PrimeScript RTase RNA cDNA . TaKaRa Ex Taq HS PCR . RNA cDNA Random 6 mers Oligo dT Primer .. 4 RR014A. IV . RNA . 12 kb . PrimeScript RTase 42 . DNA Polymerase TaKaRa Ex Taq HS. RNase Inhibitor.

5 Random 6 mers 1st strand cDNA. Oligo dT Primer .. V .. 1 PCR 10 Master Mix . Master Mix .. 2 PrimeScript RTase RNase Inhibitor TaKaRa Ex Taq HS .. 50% .. 3 . 20 20 . 4 . Positive Control RNA . 70 80 . 5 .. Random 6 mers . Oligo dT Primer 3 . mRNA 3 .. Oligo dT Primer Poly A tail mRNA . RNA rRNA tRNA mRNA poly A . tail . Random 6 mers RNA RNA . rRNA mRNA tRNA RNA . PCR .. 5 RR014A. VI . A RNA . A-1 .. dNTP Mixture 10 mM each 1 l Oligo dT Primer M . or Random 6 mers 20 M 1 l or Specific Primer 2 M 1. Template RNA 2. or Positive Control RNA 4 105 copies.

6 RNase Free dH2O up to 10 l 1 Oligo dT Primer Random 6 mers . Control RNA R-1 Primer .. 2 Template RNA 8 l total RNA 5 g 100 pg 1 g . A-2 .. 65 5 min. 4 .. RNA . RNA . A-3 .. A-2 10 l 5 PrimeScript Buffer 4 l RNase Inhibitor 40 U/ l l PrimeScript RTase for 2step l RNase Free dH2O 5 l total 20 l A-4 . 30 10 min. 3. 42 50 15 30 min. 95 5 min. 4. 4 . 3 Random 6 mers . Random 6 mers 42 50 RNA .. 4 1st cDNA . 70 15 min .. PrimeScript RTase 42 .. 50 . 6 RR014A. B PCR . B-1 .. 10 PCR Buffer II 5 l 1 . dNTP Mixture 10 mM each 2 l 400 M.

7 Primer 20 M 5 l M. Primer 20 M 6 l M. TaKaRa Ex Taq HS 5 U/ l l A-4 5 l up to 50 l 5 Positive Control RNA F-1 Primer 6 Positive Control RNA R-1 Primer B-2 PCR .. A 3 step PCR B 2 step PCR . 94 30 sec. 98 10 sec. 55 65 30 sec. 30 cycles 30 cycles 68 1 min. / kb 72 1 Positive Control RNA 7. 94 30 sec. 60 30 sec. 30 cycles 72 1 min 7 Oligo dT Primer Random 6 mers R-1 Primer . Control Primer F-1 R-1 PCR 462 bp . VII PCR .. RNA 60 PCR . 55 65 45 . 65 .. TaKaRa Ex Taq HS 72 1 kb 1 .. cDNA 40 50 cycles . PCR 3' A 1 . PCR T-Vector T-Vector Mighty TA-cloning Kit 6028.

8 Mighty Cloning Reagent Set (Blunt End) . 6027 . 7 RR014A. VIII . 1 total RNA HL60 total RNA . RT-PCR . total RNA 1 g/20 l RT 2 l 50 l PCR . total RNA 100 ng / 50 l PCR . Target gene total RNA. Dystrophin . TFR HL60 . PCR . 6 kb 8 12 kb 94 1 min. 94 1 min.. 94 30 sec. 98 10 sec. 55 30 sec. 30 cycles 30 cycles 68 8 min. or 15 min. 72 1 min. /kb M1 1 2 3 4 M2 M1 5 6 7 8 9 10 M2. M1 pHY Marker 1 TFR kb 2 TFR kb 3 TFR 3 kb 4 TFR kb 5 Dystrophin 1 kb 6 Dystrophin 2 kb 7 Dystrophin 4 kb 8 Dystrophin 6 kb 9 Dystrophin 8 kb 10 Dystrophin 12 kb M2 -Hin d III digest 12 kb.

9 8 RR014A. 2 . HL60 total RNA Oligo dT Primer GAPDH .. GAPDH 428 bp PCR 94 1 min.. 94 30 sec. 55 30 sec. 40 cycles 72 1 min. M 1 2 3 4 5 M. RNA template (total RNA). 1 pg 2 1 pg 3 10 pg 4 100 pg 5 1 ng M 100 bp Ladder total RNA 1 pg . IX RNA . RNA cDNA cDNA . RNA RNase . RNase . RNA RNase . RNA .. 1 DEPC 37 12 . 2 DEPC 120 30 . RNase RNase-OFF 9037 .. RNA RNA .. RNA . RT-PCR RNA RNA . GTC . RNA . total RNA . NucleoSpin RNA II AGPC RNAiso Plus 9108/9109 . 9 RR014A. X . PrimeScript Reverse Transcriptase 2680A/B/C . PrimeScript One Step RT-PCR Kit RR055A/B.

10 PrimeScript One Step RT-PCR Kit (Dye Plus) RR057A/B . PrimeScript High Fidelity RT-PCR Kit R022A/B . PrimeScript II High Fidelity RT-PCR Kit R023A/B . PrimeScript II High Fidelity One Step RT-PCR Kit R026A/B . PrimeScript 1st strand cDNA Synthesis Kit 6110A/B . PrimeScript II 1st strand cDNA Synthesis Kit 6210A/B . TaKaRa PCR Thermal Cycler Dice Gradient TP600 . Agarose L03 TAKARA 5003/5003B . NuSieve 3:1 Agarose 50091/50090/50094). RNase-OFF RNase 9037 . NucleoSpin RNA II . RNAiso Plus 9108/9109 . Mighty TA-cloning Kit 6028.