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1 VOLUME 2 N0. 3 Index PCR PCR TP Interview FQ
2 PCR Cloning PCR Problem PCR TaqPol I 3' T BluntPCR Solution StrataClone TM Blunt PCR Cloning Kit I Cre PCRPfu3'5' proofreading PCR StrataClone TM StrataClone TM Blunt PCR Cloning KitVaccinia DN I P1 Cre StrataClone TM Vector MixDN loxp 1PCRVector Mix DN I PCR DN 5 DN Cre SoloPack PCR Cloning Competent Cells DNloxP CreloxP lac Z 10kbPCR 95 Eco RI M13 StrataClone TM StrataClone TM TBlunt PCR TEasy- High-Fidelity PCR Cloning Enzyme# ' Taq 6 6kb PCR loxp 1 PCRStrataClone TM Vector Mix5 puc ori P lac lac Z' Topoisomerase I PCR Product lac Z' Topoisomerase I ampicillin loxp StrataClone TM Blunt PCR Cloning Kit BluntPCR Taq20 PfuUltra TM II Fusion HS DN Polymerase # PCRHerculase II Fusion DN Polymerase# PCR DN Fusion PCRPCR 1/5 PCR4050µl20 StrataClone Ultra TM Blunt PCR Cloning Kit # Cre StrataClone Competent Cells puc ori <loxp> P lac lac Z' StrataClone PCR Cloning Vector psc-b f1 ori PCR Product lac Z' ampicillin StrataClone TM PCR Cloning Kits Blunt/T # PCR&PCR StrataClone Ultra TM Blunt PCR Cloning Kit Blunt 20 (PCR ,200 PCR StrataClone TM Blunt PCR Cloning Kit Blunt ,000 StrataClone TM PCR Cloning Kit T T ,000 PCR PfuUltra TM II Fusion HS DN Polymerase Blunt ,400 Herculase II Fusion DN Polymerase Blunt ,200 Easy- High-Fidelity PCR Cloning Enzyme T 100U ,600 Strategies Vol.2 No.3 p1
3 PCR Enzymes & Instruments PCR Problem PCR Solution Herculase II Fusion DN Polymerase PCRPCR DMSO PCR PCR Herculase II Fusion DN Polymerase proofreadingpcrαpfu DN FusionPCRDN DN DN processivity Pfu proofreadingpcr 1kb12 Herculase II Fusion DN Polymeraseprocessivity 1kb15 10kb kb Template length 6 kb 12 kb min Total protocol length Fusion PfuUltra II & Herculase II Common proofreader Taq 1344 Herculase II & PfuUltra TM II Fusion Proof-ReadingTaq 2.6Kb 6Kb12Kb30 3-PCR kb ng 2 Herculase II Enzyme Ph/iP 50 Pl K Ha1T3.9kb Herculase II processivityrchaemaxx Factor 3 12kb 6 kb Herculase II Enzyme Ph/iP 50 W Seconds / kb kb6kb Herculase II15/kb GC rchaemaxx Factor GC 4 DMSO 4 Pfu Taq6 PfuUltra TM II Fusion HS DN Polymerase Taq202 ccuracy (x10 5 ) 5 Herculase II Enzyme Ph/iP PGC IGFB FMR1 HTR MMZ5 IGFB FMR1 HTR MMZ5 IGFB FMR1 HTR MMZ GC IGFB: Human Insulin-like Growth Factor (79% GC, 250bp)FMR1: Fragile Mental Retardation Syndrome protein (84% GC, 300bp)HTR: Hydroxytryptomine Receptor C2 fragment (65% GC, 540bp)MMZ5: Zinc Family Member 5 protein (68% GC, 562bp) Herculase II84% GC 0.00 PfuUltra II Polymerase ccuracy of Proofreading DN Polymerase Herculase II Polymerase Ph DV V K PU Taq Cline, J. et al. (1996) Nucleic cid Research, Vol.24,No.18: processivity rchaemaxx Polymerase Enhancing Factor Herculase II Fusion DN Polymerase # Herculase II Fusion DN Polymerase 100U ,200 dntps 500U ,600 dntps 1,000U ,600 Strategies Vol.2 No.3 p2
4 Functional nalysis Vectors TP Problem FLGGST TP Solution InterPlay TM denoviral TP System deasy TM XL denoviral Vector SystemTandem ffinity Purification (TP) InterPlay TM denoviral TP System TPInterPlay TM Mammalian TP SystemTP 2 SBP CBP 1 SBP CBPEGT InterPlay TM denoviral TP Systems InterPlay TM Mammalian TP SystemsTP deasy TM XL denoviral Vector System pfu/ml deasy TM XL denoviral Vector System ptp shuttle vector6.6kb DN epichromosomal D-293 Pac I Pme I RITR Ori TP Shuttle Vector RITR Pme I BJ5183-D-1 Kanr Ori Kanr Kanr TP Shuttle Vector Pac I Ori Pac I pdeasy-1 Vector mpr Pac I LITR Pac I LITR Encapsidation signal (ES) ES poly poly Pac I CMV LITR promoter poly denoviral DN RITR Pac I ES Left rm Left rm Right rm Right rm D-293 Pac I PROTEIN OF INTEREST WITH INTERCTING PRTNER STREPTVIDIN-BINDING PEPTIDE (SBP) CLMODULIN-BINDING PEPTIDE (CBP) CONTMINNTS STREPTVIDIN RESIN CLMODULIN RESIN 2 InterPlay TM denoviral TP System 1 TP InterPlay TM denoviral TP System # InterPlay TM denoviral N-Terminal TP System N# ,000 InterPlay TM denoviral N-Terminal TP Vectors N 116,000 InterPlay TM denoviral C-Terminal TP System C# ,000 InterPlay TM denoviral C-Terminal TP Vectors C 116,000 Strategies Vol.2 No.3 p3
5 Functional nalysis Vectors Problem CsCl Solution deasy TM Virus Purification Kit 23 deasy TM Viral Titer Kit CsCl 1248 deasy TM Virus Purification Kit 23 3 Benzonase 0.45µm Filter Sartbind Filter Unit 60ml /ml0.025 EU (endotoxin units)/ml CsCl deasy TM Viral Titer Kit (#972500) deasy TM Virus Purification Kit (#240243): ml (#240244): ml (#240245): 500ml 1 Masterflex L/S 16-size tube Peristaltic pump TCID50 (50% Tissue Culture Infectious Dose) 10 deasy TM Viral Titer Kit D-293HEK293 HEK293 D % HRP DBdiaminobenzidine HRP 1 pshuttle-ires-hrgfp-1 pshuttle-ires-hrgfp-2 24 well 120 well 1 deasy TM denoviral Vector Systems # deasy TM Virus Purification Kit 2100ml cultures , ml cultures , ml cultures ,000 deasy TM Viral Titer Kit 120wells (24well) ,000 Strategies Vol.2 No.3 p4
6 Competent Cells Problem Solution rcticexpress TM Competent Cells Oleispira antarctica Cpn60 Cpn10 E. coli Inclusion bodies in vitro refolding rcticexpress TM Competent CellsOleispira antarctica Cpn60 Cpn CodonPlus - - RIL pgbm RIL RP Cpn60/Cpn Temp ( C) kDa 62kDa Cpn60 49kDa 38kDa X-21p 28kDa 18kDa 14kDa Cpn rcticexpress TM CodonPlus 2. rcticexpress TM CodonPlus -RIL 3. rcticexpress TM CodonPlus pgbm 4. BL21-CodonPlus -RIL 5. rcticexpress TM CodonPlus -RP X-21p 1330 Cpn60 Cpn1013 X-21p CodonPlus pgbmtrn RIL/RP rcticexpress TM Competent Cells rcticexpress TM Competent Cells trn CodonPlus RILRPRI LP TRILGC RPCodonPlus StrategiesVol.2 No.2p1314 CodonPlus 2 lac UV5 T7 RN DE3 (DE3) pett7 T7 VariFlex TM SET Solubility Enhancement Tags SBPStreptavidin Binding Peptide Tag pet SET VariFlex TM StrategiesVol.2 No.2p12 CodonPlus # rcticexpress TM Competent Cells T ml ,600 rcticexpress TM (DE3) Competent Cells T ml ,600 rcticexpress TM (DE3) RIL Competent Cells T7 RILT100.1ml ,600 rcticexpress TM (DE3) RP Competent Cells T7 RPGC100.1ml ,600 rcticexpress TM RIL Competent Cells T7 RILT100.1ml ,600 rcticexpress TM RP Competent Cells T7 RPGC100.1ml ,600 Strategies Vol.2 No.3 p5
7 Cell Biology Problem Solution SatisFection TM Transfection Reagent SatisFection TM Transfection Reagent 1 DN endocytosis DN ph 1 1 SatisFection TM Transfection Reagent 208F DOY NCIH DU-145 NIH3T3 549 HeLa PC-3 B16-F0 HepG2 PtK2 B16-F10 HT-1080 RPMI-1846 C6 HUVEC SK-OV-3 CaCO-2 LnCap SVEC ChoK1 MCF-7 T24 Cos-7 MD-MB-231 U-2 OS CT26WT MDCK WEHI CV1 MS1 SatisFection TM Transfection Reagent primary cells HUVECHepG2 SatisFection TM Transfection Reagent 31 70% DN DN65% B C 1 D293pExchange-luciferase 24 D293BCSatisFection TM Transfection Reagent SatisFection TM C B SatisFection TM Transfection Reagent # SatisFection TM Transfection Reagent 0.3ml , ml , ml ,800 Strategies Vol.2 No.3 p6
8 V Tech Talk Tech Talk Tech Talk DNsiRN V 1 1 MMLV V RN DN DN 10,690 36kb 5,000 9kb 8kb 4.5kb ViraPort Retroviral Gene Expression System deasy TM denoviral Vector System V Helper-Free System gag ψ + splice donor pfb-neo-lacz Control Vector splice acceptor 5 LTR lacz transcription initiation IRES hrgfp Luc amp neo pfb-neo Cloning Vector pfb-cloning Vector IRES pfb-hrgfp Control Vector pfb-luc Control Vector pfb Retroviral Vectors neo 3 LTR pbr322 ori BGH p hrgfpβ- cis trans 3 hisd amp puc ori pvpack-mpho IRES 101 pvpack-101 mpho Eco pvpack-eco VSV-G pvpack-vsv-g pvpack-env puc ori amp pvpack-gp BGH p gag-pol IRES puro SV40 p lacz pshuttle-cmv-lacz Vector 3x H SV40 p IRES hrgfp pshuttle-ires-hrgfp-2 Vector 3x FLG SV40 p IRES hrgfp pshuttle-ires-hrgfp-1 Vector SV40 p Pac I kan L-ITR pbr322 ori pshuttle-cmv Vector pshuttle Vector ES deasy pshuttle Vectors Pac I R-ITR Pme I left arm homology right arm homology d5 (E1/E3-deleted) pdeasy-1 Vector right arm homology pbr322 ori left arm homology E. coli TP amp β-globin intron 3x FLG IRES hrgfp pv-ires-hrgfp Cloning Vector β-globin intron hghp pv- Cloning Vector β-globin intron L-ITR hgh intron hrgfp pv-hrgfp Control Vector pv-lacz Control Vector puc ori lacz pv Vectors amp hgh p SV40 p f1 ori R-ITR hghp V phelper Strategies Vol.2 No.3 p7 amp puc ori E4 a f1 ori f1 ori phelper amp f1 ori pcmv- PUC ori puc ori β-globin intron E2 p V-RC 7.3 k b hgh p V-2 cap V-2 rep
9 1 DN/RN/ RNDN nmnm1 36 RN0.1µm 4060nm RN DN MMLV : Molony Murine Leukemia Virus Oncovirus LentivirusSpumavirus3 MMLV 1 LTRLong Terminal Repeat gagpolenv5'ltrgaggag LTRDN /gagpol env RN DN DN 7090nm F6BE DFB11 E1E1BE1E1B 293 E1 E1E1BE3 8kb V ParvoviridaeDependovirus DN DN 20nm32 20 parvus V 100 V85V Tel: jtech@agilent.com Strategies Vol.2 No.3 p8
10 Cell Biology Problem -EMS Solution PathDetect Pathway Profiling Systems PathDetect PathDetect Pathway Profiling Systems PathDetect Pathway Profiling Systems cis- trans - 2Reporting System PathDetect cis-reporting Systems hrgfp in vivo in vitro PathDetect trans-reporting Systems PathDetect Cis-Reporting Systems MP MP c-jun/c-fosp-1elk-1 Egr-1NF-kBSRE JK/STT STT GSISRELILRE PathDetect cis-reporting Systems MPJK/ STT DR1/RXREDR3/VDR DR4DR5/RRE 1 PathDetect cis-reporting Systems PathDetect hrgfp pluc- plasmid PathDetect cis- 1 cis- + Cotransfection of genes TTT PO4 T PO4 T PO4 T PK POL II COMPLEX OH T + TTT 1 + Cis + + PK + trans-activatort + T Cis PathDetect Cis -ReportingSystems 1 PathDetect Cis-Reportor Plasmids P-1 (ctivator protein-1) c/ebp (CCT-enhancer binding protein) CRE (Cyclic MP) DR1/RXRE (Retinoic x receptor epsilon) DR3/VDR (Vitamin D receptor) DR4/TR (Thyroid receptor) DR5/RRE (Retinoic acid receptor epsilon) Egr-1 (Early growth response-1) GS (g-activated sequence) GRE (Glucocorticoid response element) ISRE (Interferon-stimulated response element) LILRE (LPS IL-1b response element) NFT (Nuclear factor of activated T cells) NF-kB (Nuclear factor kb) p53 (Tumor suppressor protein p53) SRE (Serum response element) SRF (Serum response factor) TRE (TGF-Beta or ctivin response element) Strategies Vol.2 No.3 p9
11 Northern & Southern nalysis Problem Solution Prime-It II Random Primer Labeling Kit dpm/µg 2 2 [ 32 p]dtp[ 32 p]dctp 9mer Prime-It RmT Random Primer Labeling Kit DN I 6 5 in situ FISH Prime-It Fluor Fluorescence Labeling Kit9-mer exo- Klenow fluor-12-dutp DN 2 NucTrap Probe Purification ColumnsG-50 DNRN 99% 17mer701750mer 80%51mer90% Push Column Beta Shield DeviceRI NucTrap Probe Purification Columns cm3 12 QuikHyb Hybridization Solution 2 MiracleHyb TM Hybridization Solution RN 24 # QuikHyb Rapid Hybridization Solution 1,000ml , ml ,000 MiracleHyb TM Hybridization Solution 125ml , ml ,000 Salmon Sperm DN (10µg/ml) 10ml ,000 Prime-It Fluorescence Kit ,600 Prime-It II Random Primer Labeling Kit ,600 Prime-It RmT Random Primer Labeling Kit ,800 NucTrap Probe Purification Columns , ,600 Push Column Beta Shield Device ,800 Push Column Extra Syringe, ,000 Strategies Vol.2 No.3 p10
12 Q. Lead Vocal Vocalist Vocalist Vocalist 20 Mx3000P PCR Multiplex Multiplex Multiplex NIH HCMV HHV-6EBVKeynotes Mx3000P PCR Keynotes Herpusvirus DN DN150kb80 ORF nm nm 130 αβhcmvhhv-6 γebv B HCMV: Human Cytomegalovirus 235kbDN180nm 0.4-1%5-10%10% 6HHV-6: Human Herpesvirus 6 160kbDNBHHV-6 HHV6HHV-6B 1 EBEBV: Epstein-Barr Virus 172kbDN HHV4 90% B EBVBTNK Strategies Vol.2 No.3 p11
13 Q. Mx3000P PCR. MultiplexMx3000P PCR Q.. Keynotes3 Q.. 3 Herpes-Simplex Virus 5 Mx3005P 2 Mx3005P Mx3000P PCR Q. 54PR Q. Multiplex. Multiplex &Overview & Real-time PCREBV DN Kimura, J. Clin Microbiol Multiplex real-time PCR EBV, CMV, HHV6 EBV BLF5-FM CMV IE2-HEX HHV-6 U31-Cy5 54http://virus54.umin.jp/ Tel Fax Strategies Vol.2 No.3 p12
14 FQ deasy TM deasy TM denoviral System TP Q deasy TM denoviral Vector System deasy TM XL denoviral Vector System InterPlay TM denoviral TP System Q deasytm denoviral Vector SystemdEasy TM XL denoviral Vector System deasy TM denoviral deasy TM denoviralpshuttle pdeasy-1e. coli BJ5183 E. coli InterPlay TM denoviral N-Terminal TP System N# ,000 N-Terminal TP Vectors N 116,000 C-Terminal TP System C# ,000 C-Terminal TP Vectors C 116,000 deasy TM Virus Purification Kit 2x x 100ml cultures 94,000 5x x 100ml cultures 208, x 500ml cultures 188,000 deasy TM Viral Titer Kit wells (24well) 92,000 deasy TM denoviral Vector System # ,000 BJ5183 Electroporation-Competent Cells µl 49,600 pdeasy-1 vector µg 82,000 pshuttle vector µg 82,000 pshuttle-cmv vector CMV20µg 82,000 pshuttle-cmv-lacz vector LacZ 10µg 66,000 pshuttle-ires-hrgfp-1 vector FLGhrGFP20µg 104,000 pshuttle-ires-hrgfp-2 vector H taghrgfp20µg 104,000 D-293 cells ,000 deasy TM XL denoviral System #200157# ,000 BJ5183-D-1 Electroporation-Competent Cells µl 65,200 Strategies Vol.2 No.3 p13
15 Q deasytm denoviral Vector SystemdEasy TM XL denoviral Vector System deasy TM denoviralpshuttle pdeasy-1e. coli BJ5183 deasy TM XL denoviraldeasy-1 BJ5183-D1 pshuttle2 BJ51833 Q InterPlayTM denoviral TP System InterPlay TM denoviral TP SystemdEasy TM XL denoviral Vector SystemTandem ffinity Purification System (TP) Strategies Vol.2 No.3 P3 Q pshuttle deasy TM denoviralpshuttle7.5kb pshuttle-cmv6.6kbpshuttle-ires-hrgfp-1 pshuttle-ires-hrgfp-25.2kb InterPlay TM denoviral TP ptp Shuttle6.6kb Q deasytm denoviralinterplay TM denoviral TP D-293 Q D-293 ViraPack Transfection Kit (#200488) 10 7 pfu/ml Q 1248dEasy TM Virus Purification Kit 23 Strategies Vol.2 No.3 P4 Q 50% Tissue Culture Infectious Dose (TCID50) 10 deasy TM Viral Titer Kit2448 pshuttle-ires-hrgfp-1pshuttle-ires-hrgfp-2 Strategies Vol.2 No.3 P4 D-293HEK293 D-293 Strategies Vol.2 No.3 p14
16 rcticexpress TM 30%OFF!! # rcticexpress TM (DE3) RIL Competent Cells 100.1ml ,500 29,750 rcticexpress TM (DE3) RP Competent Cells 100.1ml ,500 29,750 rcticexpress TM RIL Competent Cells 100.1ml ,500 29,750 rcticexpress TM RP Competent Cells 100.1ml ,500 29,750 rcticexpress TM Competent Cells 100.1ml ,500 29,750 rcticexpress TM (DE3) Competent Cells 100.1ml ,500 29, VariFlex BL21 Competent Cells30%OFF!! # BL21 Competent Cells 50.2ml ,300 20,500 BL21(DE3) Competent Cells 50.2ml ,300 20,500 BL21(DE3) plyss Competent Cells 50.2ml ,300 20,500 BL21-CodonPlus (DE3)-RIL Competent Cells 100.1ml ,600 29,100 BL21-CodonPlus (DE3)-RILP Competent Cells 100.1ml ,600 29,100 BL21-CodonPlus (DE3)-RIL-X Competent Cells 100.1ml ,600 29,100 BL21-CodonPlus (DE3)-RP Competent Cells 100.1ml ,600 29,100 BL21-CodonPlus (DE3)-RP-X Competent cells 100.1ml ,600 29,100 BL21-CodonPlus -RIL Competent Cells 100.1ml ,600 29,100 BL21-CodonPlus -RP Competent Cells 100.1ml ,600 29,100 BL21-Gold (DE3) Competent Cells 100.1ml ,000 23,800 BL21-Gold (DE3)pLysS Competent Cells 100.1ml ,000 23,800 BL21-Gold Competent Cells 100.1ml ,000 23,800 VeriFlex TM N-Terminal SBP Vector Set ,000 79,100 VariFlex TM N-Terminal SBP System , ,200 VariFlex TM N-Terminal SBP-SET Vector Set , ,000 VariFlex TM N-Terminal SBP-SET Expression System , ,200 VariFlex TM N-Terminal SET Vector Set , ,100 VariFlex TM C-Terminal SBP Vector ,000 60,200 VariFlex TM C-Terminal SBP System , ,800 VariFlex TM C-Terminal SBP-SET Vector Set , ,800 VariFlex TM C-Terminal SBP-SET Expression System , ,300 VariFlex TM C-Terminal SET Vector Set , , jtech@stratagene.com U R L
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JPN Strategies VOLUME 2 N0. 1 Incubate PCR product with Topoisomerase I charged vector arms Topoisomerase I Index 1 Fusion DN 2 3 3 PCR 4 5 RN StrataClone Competent Cells with Cre Recombinase 6 Universal
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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
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Strategies VOLUME 1 中綴じ付録 両面ポスター 遺伝子型や株の由来等の情報が一目で分かるストラタジーンのコンピテント セル一覧表 正確性や用途等の情報が一目で分かるストラタジーンのPCR酵素と逆転写酵素一覧表 ページ Index 1 サンプルのロスを最小限に抑えて より正確な発現解析を実現 2 定量PCR用に最適化した高感度の cdn合成キット 3 高品質でそのまま実験に使用できる
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D = [a, b] [c, d] D ij P ij (ξ ij, η ij ) f S(f,, {P ij }) S(f,, {P ij }) = = k m i=1 j=1 m n f(ξ ij, η ij )(x i x i 1 )(y j y j 1 ) = i=1 j
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４章 困難な課題への挑戦 核酸結晶学
4-2-6. I. a. DNA DNA RNA Watson & Crick 1953 b. DNA DNA DNA DNA RNA Cech et al. 1981 RNA in vitro selection DNA Breaker & Joyce 1994 2% 50% 30% International Human Genome Sequencing Consortium 2001 1 DNA
1 (Berry,1975) 2-6 p (S πr 2 )p πr 2 p 2πRγ p p = 2γ R (2.5).1-1 : : : : ( ).2 α, β α, β () X S = X X α X β (.1) 1 2
2005 9/8-11 2 2.2 ( 2-5) γ ( ) γ cos θ 2πr πρhr 2 g h = 2γ cos θ ρgr (2.1) γ = ρgrh (2.2) 2 cos θ θ cos θ = 1 (2.2) γ = 1 ρgrh (2.) 2 2. p p ρgh p ( ) p p = p ρgh (2.) h p p = 2γ r 1 1 (Berry,1975) 2-6