PCR Enzymes & Instruments PCR Stratagene Gradient Cycler Problem PCRDNA Polymerase Solution Stratagene Gradient Cycler PCRQuikChange 3 Stratagene Gradient Cycler 96 96 Fast 384 3 3 0.4 0.1 12 96 Fast 384 5 / 3.5 / Stratagene Gradient Cycler AT GC USB 4PCR PfuTurbo PfuUltra II Fusion Herculase II Fusion Paq5000 3 QuikChange 7 2 Stratagene Gradient Cycler NIST GLP Stratagene Gradient Cycler2 Stratagene Gradient Cycler 96 96 Fast 384 # G5100A G5100B G5100C 980,000 980,000 980,000 # G5100-64001 G5100-64002 G5100-64003 310,000 430,000 430,000 3 /2 / 5 /3.5 / 5 /3.5 / 4 4 4 30 99 30 0.4 at 95 0.1 at 95 4 99 111 120 1 1 6.5 in. (16.5 cm) LCD 10,000 7 USB USB PC Windows XP GLP USB 2.0 USB USB 31 cm (w) x 23 cm (h) x 45 cm (d) 14 kg 90V 250V NIST 2 Strategies Vol.4 No.1 p1
PCR Enzymes & Instruments PCR Paq5000 TM Problem PCR Solution PCR Paq5000 TM Taq Pyrococcus PCR Paq5000 TM DNA Polymerase Pyrococcus PCRTaq PCR Pfu PCR 3' 5' 5' 3' Taq PCR1kb 60 1kb30 Taq PCR4kb 1kb30 6kb PCR Terminal deoxynucleotidyl Transferase (TdT) TdT3' AExo Exo 3'A DNADNA 100 1 Paq5000 TM Hotstart DNA Polymerase Taq polymerase Human α1 Antitrypsin3.9kb Paq5000 TM Hotstart DNA Polymerase 30 /kbtaq Paq5000 TM Hotstart DNA Polymerase Taq PCR Paq5000 TM Hotstart DNA Polymerase 3 #600864 (5,000U)1U 26.4 Taq PCRPCR 3.9kb Paq5000 Hotstart I I Q Q R R C C A A 1 Paq5000 TM Hotstart DNA Polymerase Hotstart Taq Human α1 Antitrypsin 3.9kb Paq5000 TM Hotstart DNA Polymerase Hotstart Taq Paq5000 TM 30 /kb Paq5000 TM Hotstart DNA Polymerase PCR DNA 95 2 Paq5000 TM DNA Polymerase # Paq5000 TM DNA Polymerase 500U 600680 11,200 1,000U 600682 16,800 5,000U 600684 50,000 Paq5000 TM Hotstart DNA Polymerase 500U 600860 28,000 1,000U 600862 44,000 5,000U 600864 132,000 Strategies Vol.4 No.1 p2
QPCR & QRT-PCR Enzymes & Instruments QPCR Brilliant II 48 FAST ROX Problem PCR Solution Brilliant II FAST Master Mix Brilliant II 40 48 Brilliant II Low/High ROX TM Master Mix ROX TM reference dye 48 Brilliant II FAST Brilliant II FAST QPCR PCR 40 48 PCR Brilliant II FAST QPCR DNADNA cdna Taq DNA polymerase Brilliant II Brilliant II FAST FASTQPCR 1 Mx3005P PCR Ct (Ct) Brilliant II 2 Brilliant II ROX Brilliant II Low/High ROX TM Master Mix PCR ROX TM reference dye Brilliant II Low/High ROX TM Master Mix ROX TM reference dye Brilliant II Low/High ROX TM Master Mix ROXLow High 2 Low ROX TM Stratagene Mx3000P Mx3005P, Mx4000P, Applied Biosystems 7500 High ROX TM Applied Biosystems ABI PRISM 7000 ABI PRISM 7700, 7300, 7900HT, 7900HT Fast 1 Brilliant II FAST FAST Ct 2 Brilliant II FAST Brilliant II SYBR Green Brilliant II ROX Low ROX High ROX ROX Low ROX High ROX QPCR Master Mix 400 600804 600806 600805 600828 600830 600829 10 x 400 600815 600817 600816 600831 600833 600832 FAST Master Mix 400 600845 600843 10 x 400 600846 600844 400 600809 600837 600838 600825 600835 600836 Master Mix QRT-PCR 10 x 400 600818 600841 600842 600826 600839 600840 1-step 400 600810 Core Reagents 10 x 400 600819 QRT-PCR 2-step 400 600827 600834 Strategies Vol.4 No.1 p3
PCR Cloning StrataClone TM Problem Solution StrataClone TM Mammalian Expression System PCR 5 5 StrataClone TM Mammalian Expression System PCR StrataClone TM Cre 2PfuUltra TM II HS DNA polymerase PCR Vector Mix 5 StrataClone TM SoloPack Competent Cells 1 CMV (CMV) kanamycin/neomycin 3FLAG c-myc C- N- 3 x FLAG 3 x c-myc N- C- 5 3 in situ 16 64 FLAG c-myc 8 DYKDDDDK 10 EQKLISEEDL StrataClone TM StrataClone TM PCR Cloning Kit StrataClone TM Blunt PCR Cloning Kit Paq5000 TM DNA Polymerase PCR6kb PCR P2 PCRStataClone Vector Mix 5 loxp puc ori PCR Product FLAG c-myc none Topoisomerase I P CMV BGH pa N-Tag C-Tag neo/kan loxp Cre StrataClone Competent Cells puc ori P CMV StrataClone Mammalian Expression Vectors N-terminal tag (if present) PCR Product C-terminal tag (if present) BGH poly A Topoisomerase I FLAG c-myc none 1 StrataClone TM PCR Cloning Kit <loxp> neomycin/kanamycin StrataClone TM StrataClone TM Mammalian Expression Untagged Vector System 20 240228 62,800 StrataClone TM Mammalian Expression N-terminal FLAG Vector System 20 240229 62,800 StrataClone TM Mammalian Expression C-terminal FLAG Vector System 20 240230 62,800 StrataClone TM Mammalian Expression N-terminal c-myc Vector System 20 240231 62,800 StrataClone TM Mammalian Expression C-terminal c-myc Vector System 20 240232 62,800 Anti-FLAG M2 Antibody, 200 µg 200µg 200471 41,600 Anti-c-Myc monoclonal Antibody (Clone 9E10) 25µg 257260 16,800 Strategies Vol.4 No.1 p4
Agilent 2100 Agilent Technologies Problem SDS-PAGE () 3 Solution Agilent 2100 Agilent High Sensitivity Protein 2504 0.01% Agilent 2100 "Lab-on-a-chip" 1999 Agilent 2100 Agilent1 DNA RNA Agilent High Sensitivity Protein 250 Agilent High Sensitivity Protein 250 BSA on-chip 0.4 pg/µl 4.2 ng/µl 1 2 ph ph8 9 primary amino 0.1M Sodium citrate ph3.0 6.0 0.1M Sodium acetate ph4 ph 50mM NaOH 2.7 pg/µl 0.09% 5.4 pg/µl 0.18% IgG 2886 pg/µl 93.5% 150 kda 1 Agilent 2100 2 (BSA) 1000, 100, 10, 1, 0.1 ng/µl 1:200 1 µg/µl 1:200 1:2,000,000 (On-chip0.4 pg/µl 4.2 ng/µl) 53kDa 59kDa 3 0.09% 1 mg/ml Agilent 2100 Agilent 2100 2007 221CFR Part11 Agilent 2100 2,901,000 Agilent 2100 3,037,000 Agilent 2100 G2948C Agilent 2100 3,346,000 Agilent 2100 3,506,000 Agilent High Sensitivity Protein 250 5067-1575 64,000 Strategies Vol.4 No.1 p5
Agilent 2100 Agilent Technologies Agilent 2100 PCR Problem RNAPCR SYBR Green I Solution Agilent 2100 RNA RIN RNA Integrity NumberRNA 30 RNA RIN (RNA Integrity Number) RNA FFPE RNA ChipAgilent 2100 RNA, RIN RNA Integrity Number RIN 8.9 0 min RIN 4.6 45 min RIN 6.5 30 min RIN 2.3 75 min 1 Total RNA 75 RIN RNA QPCR 4 RNA1 RIN2.3 4.6 6.5 8.9GAPDH HPRT1 YWHAZ3'5' PCR 2 RIN GAPDH HPRT1 3'5' YWHAZ 5'3' RNAcDNA RNA RNA A 0-2 -4-6 -8-10 -12-14 B 0-1 -2-3 -4-5 -6-7 -8-9 -10 C 0-1 -2-3 -4-5 -6 RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3 RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3 RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3 GAPDH 5 assay GAPDH 3 assay HPRT1 5 assay HPRT1 3 assay YWHAZ 5 assay YWHAZ 3 assay 2 33'5' A: GAPDH B: HPRT1 C: YWHAZ SYBR Green I SYBR Green I 32 2 2 SYBR Green I Tm SYBR Green I Tm Agilent 2100 3 Agilent 2100 Agilent 2100 Agilent 2100 marker 900, 1000 bp 473, 500 bp 300, 315 bp 100, 105 bp Agilent 2100 900, 1000 bp 473, 500 bp 300, 315 bp 100, 105 bp 4 Agilent 2100 Strategies Vol.4 No.1 p6
Agilent 2100 Agilent Technologies Quality Check Problem Total RNA UV-VIS RNA Solution Agilent 2100 RIN RNAUV RNA Total RNA Total RNA RNA RNA RNA 260nm 280nm 2 1 B RNA A B RIN Total RNA Total RNA 18S/28S rrna RNA Agilent 2100 RIN RNA Integrity Number Total RNA RNA1 10 2 RIN Total RNA rrna BMC Molecular Biology 2007.8:38 2 Total RNA RIN Total RNA Total RNA 3 Scatter Plot Total RNA RIN2.4Intact Total RNA RIN7.3 RNA A 3. A Intact Total RNA B Intact Total RNA Total RNA B 1. ARNA UV BRNA UV Agilent 2100 Agilent RNA6000 RNA252 5067-1511 71,000 Total RNA mrna RNA6000RNA Agilent RNA6000 RNA253 5067-1513 76,000 Total RNA mrna RNA6000RNA Agilent Small RNA 253 5067-1548 99,000 Small RNA Small RNASmall RNA Small RNA Ver. B.02.03 Strategies Vol.4 No.1 p9
Microarray Analysis DNA Problem Solution CGH / CNV mirna ChIP-on-chip DNA DNA DNA CGH ChIP-on-chip RNA 1 x 3 inch 60mer In situ 1 24 2008 100 100% DNA 3 log 5 log Human, Mouse, Rat 5 log CGH / CNV FFPE mirna mirna Sanger Institute mirbase 10.1 Human, Mouse, Rat 12.0 ChIP-on-chip MIT ChIP-on-chip Human Mouse DNA- Protein ( ) DNA CpGDNA Web earray 1 QC ) GeneSpring DNA Analytics QC Strategies Vol.4 No.1 p10
Microarray Analysis 5log Problem 3 log Solution 5 logpcr 3log 5log 1B 1 2 Feature Extraction 2005 FDA MAQC Microarray quality control Project PCR PCR MAQC 2 T7 RNA polymerase RNA P9 1 12 10 Agilent Gene Expression log ratio 8 6 4 2 0-2 -4-6 -8-10 -12 User1 : n=607, r =0.94, m=0.94 User2 : n=602, r =0.94, m=0.97 User3 : n=597, r =0.94, m=0.96-12 -10-8 -6-4 -2 0 2 4 6 8 10 12 qpcr log ratio 1 2 PCRPCR MAQC Strategies Vol.4 No.1 p11
Software Solutions GeneSpring GX Problem Solution GeneSprin GX GeneSpring GXmiRNAExonPCR GeneSpring GXQuality check 1 T ANOVA 2 2 Volcano plot 1 3D PCA GeneSpring GX median shift Quantile Scaling GeneSpring GX GO Analysis 3 Find Significant Pathway 4 4 Pathway View NCBI Biological Genome Homology Table ID 3 GO Pie chart Strategies Vol.4 No.1 p12
FAQ Agilent 2100 Agilent 2100 Lab-on-a-Chip Agilent 2100 RNA DNA SDS-PAGE Agilent 2100 Agilent 2100 Lab-on-a-Chip DNA RNA http://www.chem-agilent.com/contents.php?id=50 Q Agilent A 2100 Agilent 2100 1 RNA DNA Q Agilent A 2100 1 4µl301 1. 5 µl 1 2. 3. 30 / RNA QC RIN 3 Q Agilent A 2100 2 Agilent 2100 Agilent 2100 1. 2. 3. Agilent 2100 21 CFR Part11 4. 5. PC Strategies Vol.4 No.1 p13
Q Agilent A Agilent 2100 5,000 7,000 PCRRNA RNA Q A 2100 PCR/multiplex PCR RT-PCRDNA Protein Protein RNA 5067-1511 Agilent RNA6000 5067-1513 Agilent RNA6000 5067-1548 Agilent Small RNA Agilent Protein Agilent Q RIN A (RNA Integrity Number) RIN RNA 3 RNA RNA 10RNA RNA RIN PCR Agilent 2100 Agilent RNA RIN RNA DNA 5067-1504 Agilent DNA1000 5067-1506 Agilent DNA7500 5067-1508 Agilent DNA12000 Protein 5067-1517 Agilent Protein 230 5067-1515 Agilent Protein 80 5067-1575 Agilent High Sensitivity Protein 250 2 RNARIN (RNA Integrity Number) 5067-1519 Agilent Agilent RNA RIN RNA Integrity NumberRNA 25 50 total RNA mrna Agilent DNA DNA PCR RIN: 1 RIN: 2 RIN: 3 RIN: 4 RIN: 5 RIN: 6 RIN: 7 RIN: 8 RIN: 9 RIN: 10 3 RINRNA Strategies Vol.4 No.1 p14
Mx3000P QPCR 2009 3 31 Mx3000P 3,500,000 2,980,000!! RNA - Absolutely RNA Purification Kit - AffinityScript RT PCR - Brilliant Master Mix RNA - Alien Control RNA - Stratagene QPCR Reference Total RNA QPCR 2009 3 31 Brilliant II Fast 20% OFF!!!!! # Brilliant II Fast SYBR Green QPCR Master Mix 400 600843 46,000 36,800 Brilliant II Fast SYBR Green QPCR Master Mix 4,000 600844 404,800 323,840 Brilliant II Fast QPCR Master Mix 400 600845 46,000 36,800 Brilliant II Fast QPCR Master Mix 4,000 600846 404,800 323,840 2009 3 31 Agilent 2100 3,037,000 + Mx3000P PCR 3,500,000 + 4,980,000 ( 5,000 2009 3 31 Agilent 2100 Plus 2 9 2!! * 192-8510 9-1 0120-477-111 email_japan@agilent.com www.agilent.com/chem/jp