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1 JPN Strategies VOLUME 2 N0. 1 Incubate PCR product with Topoisomerase I charged vector arms Topoisomerase I Index 1 Fusion DN PCR 4 5 RN StrataClone Competent Cells with Cre Recombinase 6 Universal Reference RNs GFP 12 DN RN FQ SideStep TM Stratagene
2 PCR Enzymes & Instruments Fusion DN Problem Proof-Reading PCR Solution PfuUltra TM II Herculase II Fusion DN Polymerase Fusion DN Pfu DN DN Fusion DN DN DN Processivity rchaemaxx Factor PCR dctp dutp dutpdn Pfu Proof-Reading DN rchaemaxx Factor dutp dump DN dutp PCR Fusion DN rchaemaxx Factor PfuUltra TM II Fusion HS DN Polymerase PfuUltra TM II Fusion HS DN Polymerase PfuUltra TM High Fidelity DN Polymerase Fusion Taq20Pfu Kb Hot Start FusionHot Start Processivity Pfu 125 1Kb15 10Kb 30 ccuracy (x10 5 ) PfuUltra II Polymerase Herculase II Polymerase ccuracy of Proofreading DN Polymerase Ph DV V K PU Taq GC Herculase II Fusion DN Polymerase Herculase II Fusion DN Polymerase Pfu Taq Pfu Herculase DN Polymerase Taq6 Taq4 GC DMSO GC 84% GC Fusion Herculase II Fusion DN Polymerase 1ng15 /Kb 30 /Kb 1 Nucleic cid Research, 1996, Vol.24, No.18: Fusion DN Polymerase # PfuUltra TM II Fusion HS DN Polymerase , , ,800 Herculase II Fusion DN Polymerase , , ,600 Strategies Vol.2 No.1 p1
3 Protein Expression Vectors Problem 1 Solution pcmv-3tag Epitope Tagging Mammalian Expression Vectors 3 FLG c-myc 3 pcmv-3tag vectorn- C-FLG c-myc 3 3 FLG c-myc8 DYKDDDDK 10 EQKLISEEDL FLG /c-myc N- /C pcmv-3tag-1 4 / pcmv-3tag-6 9 (CMV) Kozak( ) N- 1 pcmv-3tag vector N- C- FLG # # c-myc # # FLG # # c-myc # # localeid=2 FLG c-myc pcmv-3tag-4(abc) TK p TK p 1 puc ori Neo/Kan Hyg puc ori P CMV pcmv-3tag-1 4 Neomycin/Kanamycin pcmv-3tag-2(abc) pcmv-3tag-1(abc) 3x-FLG BGH p pcmv-3tag-3(abc) 3x-c-Myc f1 ori pcmv-3tag-8 P bla P SV40 pcmv-3tag-7 3x-c-Myc P CMV pcmv-3tag-6 3x-FLG pcmv-3tag-6 9 Hygromycin P TK amp BGH p f1 ori pcmv-3tag-9 pcmv-3tag vector 3x-FLG 3x-FLG 3x-c-Myc 3x-c-Myc pcmv-3tag Epitope Tagging Mammalian Expression Vectors # pcmv-3tag-1 (abc) vectors 3 x 20µg ,000 pcmv-3tag-2 (abc) vectors 3 x 20µg ,000 pcmv-3tag-3 (abc) vectors 3 x 20µg ,000 pcmv-3tag-4 (abc) vectors 3 x 20µg ,000 pcmv-3tag-6 vector 20µg ,000 pcmv-3tag-7 vector 20µg ,000 pcmv-3tag-8 vector 20µg ,000 pcmv-3tag-9 vector 20µg ,000 nti-flg M2 ntibody 200µg ,600 1mg ,000 nti-c-myc Monoclonal ntibody 25µg ,800 (Clone 9E10) 100µg ,000 Strategies Vol.2 No.1 p2
4 PCR Cloning PCR Problem PCR Solution StrataClone TM PCR Cloning Kit DN 9kbPCR 3 95% PCR 20 StrataClone TM PCR Cloning Kit PCR PCR PCR 3 PCRVector Mix 5 DN 1) 9kbPCR 95 ( 1) loxp 1 PCRStrataClone TM Vector Mix 5 puc ori P lac lac Z' Topoisomerase I PCR Product U* U* 1 StrataClone TM PCR Cloning Kit PCR Insert Size (bp) 664* % 1.8K* % 3.5K* % 6.0K** % 9.2K** % *PCR2µl ** StrataClone TM VacciniaDN P1Cre DNDN Vaccinia DN DN DN DNDN DN Cre 2 8 loxp lac Z' StrataClone TM PCR Cloning Kit Topoisomerase I ampicillin loxp Cre StrataClone Competent Cells StrataClone TM Vector Mix DN loxp Taq DN PCR PCR Vector MixDN PCR DN 5 DN Cre SoloPack PCR Cloning Competent Cells DNloxP Cre loxp lacz Eco RI M13 puc ori <loxp> P lac lac Z' StrataClone PCR Cloning Vector psc- PCR Product lac Z' ampicillin StrataClone TM PCR Cloning Kits # StrataClone TM PCR Cloning Kit ,000 Strategies Vol.2 No.1 p3
5 Competent Cells Problem Solution Protein Expression Competent Cell Pack 4 BL21 (DE3) Competent Cells T7 BL21-CodonPlus (DE3)-RIPL Competent Cells4 R I P L 1 BL21-Gold (DE3) plyss Competent Cells T7 T7 RN BL21-Gold (DE3) Competent Cells End 1 Difficult Cloning Competent Cell Pack 3 Hte XL10-Gold Kan r Ultracompetent Cells Inverted RepeatZ-DN SURE 2 Supercompetent Cells 2 ColE-1 BLE K Competent Cells DN hctnt BL21(DE3) cells BL21-CodonPlus (DE3)-RIPL cells CBP-3xl-Cre BL21(DE3) cells BL21-CodonPlus (DE3)-RIPL cells CBP-3xP-Cre BL21(DE3) cells BL21-CodonPlus (DE3)-RIPL cells 1 BL21-CodonPlus (DE3)-RIPL Competent Cells BL21 (DE3) Competent Cells BL21-CodonPlus (DE3)-RIPL Competent Cells T GC hctnt T argu CBP-3xl-Cre ilew CBP-3xP-Cre prol Routine Cloning Competent Cell Pack 3 XL2-Blue MRF' Ultracompetent Cells K12 Competent Cell Packs DN XL2-Blue Ultracompetent Cells DN 5x10 9 SoloPack Gold Competent Cells XL10-Gold β- SURE cells DH5α HB101 SURE cells DH5α HB % Z-DN % 2 SURE DN Protein Expression Competent Cell Pack ,000 BL21-CodonPlus (DE3)-RIPL Competent Cells 2 x 0.1ml BL21-Gold (DE3) plyss Competent Cells 2 x 0.1ml BL21-Gold (DE3) Competent Cells 2 x 0.1ml BL21 (DE3) Competent Cells 1 x 0.2ml Difficult Cloning Competent Cell Pack ,000 XL10-Gold Kan r Ultracompetent Cells 2 x 0.1ml SURE 2 Supercompetent Cells 2 x 0.1ml BLE K Competent Cells 1 x 0.2ml Routine Cloning Competent Cell Pack ,000 XL2-Blue Ultracompetent Cells 2 x 0.1ml XL2-Blue MRF' Ultracompetent Cells 2 x 0.1ml SoloPack Gold Competent Cells 2 x 0.025ml Strategies Vol.2 No.1 p4
6 Nucleic cid Sample Preparation RN Problem RN (mirn) mirnrn mirn Solution mircle TM mirn Isolation Kit mirn 30QPCR mirn mirn mirnmirn Total RN Lysis Buffer DNRN mircle TM mirn Isolation Kit mirntotal RN mirn PCR mirn DN SPIN SPIN mirn let7d mirn RN PCR ( 3) Mx3000P QPCRBrilliant SYBR Green QPCR mirn ( 1) RN 1 35%(V/V) - PCR (QPCR) let7d mirnin vitro let7d mirn 30 RN-Binding Spin Cup RN SPIN SPIN RN (10 200b) Mouse Brain 44mg 2.2µg Mouse Kidney 127mg 5.9µg Mouse Liver 146mg 13.1µg Mouse Testes 42mg 1.8µg Rat Lung 50mg 3.5µg HeLa S µg Human Placenta 36mg 1.3µg 1 mirn 5 150mg %(V/V) RN-Binding Spin Cup RN Low-Salt Wash Buffer RN 3 SPIN SPIN 5 150mg mirn RN50 Fluorescense (drn) Mouse brain Mouse kidney Human placenta Mouse testes Mouse liver Rat lung HeLa S3 No QPCR primers Cycles RN SPIN SPIN 2 1 mirn let7d mirn 1 mircle TM mirn Isolation Kit mircle TM mirn Isolation Kit # mircle TM mirn Isolation Kit , ,200 Strategies Vol.2 No.1 p5
7 Microarray nalysis Universal Reference RNs Problem Reference RN Solution Universal Reference RN Universal Reference RN Universal Reference RN Total RN 1 Universal Reference RN 3 Universal Human Reference RN gilent Bioanalyzer 18S 28S rrn RIN Human Cell Lines Mouse Cell Lines Rat Cell Lines Brain Breast B-Lymphocyte Cervix Liver Liposarcoma Macrohages Skin Testis T-Lymphocyte 1 Universal Human Reference RN B-Lymphocyte Embryo (2) Kidney Liver Macrohages Mammary Gland Muscle Skin Testis T-Lymphocyte Universal Mouse Reference RN Universal Reference RN RN Universal Reference RN Basophile B-Lymphoblast Brain Embryo (2) Kidney Liver Lung Macrohages Mammary Gland Muscle Skin Testis T-Lymphocyte Universal Rat Reference RN Universal Reference RN 2 RN RN 3 Tested RN 1 URR Universal Reference RN (URR) Tested RN 2 URR Tested RN 3 Labeling Reaction Cy5 Cy3 Cy5 Cy3 Cy5 Cy3 Cy5 Cy3 Tested cdn Reference cdn Tested cdn Reference cdn Microarray Hybridization Data nalysis Tested cdn Strategies Vol.2 No.1 p6 URR Reference cdn Tested RN 4 Tested cdn rray 1 rray 2 rray 3 rray 4 Sample 1 Sample 2 Sample 3 Sample 4 Reference Reference Reference Reference Reference cdn 3 Universal Reference RN Two-Hybridization 28S/18S ratio = 1.7 RIN = 9.3 Universal Reference RN # Universal Human Reference RN 2 x 200µg ,600 Universal Mouse Reference RN 2 x 200µg ,600 Universal Rat Reference RN 2 x 200µg ,600
8 Tech Talk Tech Talk Tech Talk PCR DN RN 260nm (OD260)DN RN DN RN Blank Spectrophotometric Conversions for Nucleic cids 260 = 1 (1cm) Concentration (µg/ml water) ds DN 50 ss DN 33 ss RN 40 Oligonucleotide DN DN 50µl cm length) DN 50µg/ml x 260 x 50µg/ml x x µg/ml OD260 OD260OD /280 phph 260/ /280 DN10mM Tris ph RN10mM Tris ph nm 270nm 325nm 1 330g/mol 17mer 330g/mol x 17bases = 5610g/mol 1,000bp DN 1µg 1.52pmol DN 3.03pmol RNDN RN RN Qanti-It TM RiboGreen RN Mx3000P / Mx3005P PCR PCR Quanti-It TM RN DN x DN 1875µg/ml x 0.050ml 93.75µg Mx3000P Mx3005P Quanti-It TM RiboGreen Strategies Vol.2 No.1 p7
9 Topoisomerase I Mx3000P /Mx3005P PCR 16S23S rrn ng 0.5ng/µl 1µl 25µl 20pg/µl RiboGreen RN RiboGreen 2 RiboGreen RiboGreen Range of RN amounts dilution factor minimum maximum 1:400 5ng 100ng 1: ng 10ng RN 25µl 50ng/µl 40ng/µl 30ng/µl 20ng/µl 10ng/µl 5ng/µl 2ng/µl 1ng/µl 0ng/µl 5ng/µl 4ng/µl 3ng/µl 2ng/µl 1ng/µl 0.5ng/µl 0.1ng/µl 0ng/µl Mx3000P PCR Mx3000P MxPro TM Quantitative Plate Read RN (ng) mean RFUs σ RN (ng) mean RFUs σ MxPro TM nalysis y=mx+b) RSq RSq Mx3000P /Mx3005P PCR PCR SNP RN DN OD260nm Mx3000P /Mx3005P PCR Mx3005P FRET 25 FM/SYBR 492nm 516nm Mx3000P PCR Cat# Mx3005P PCR Cat# Strategies Vol.2 No.1 p8
10 Nucleic cid Sample Preparation Problem (FFPE) PCR RN RN Solution bsolutely RN FFPE Kit RN RN PCR RN RN FFPE QRT-PCR mrn QRT-PCR RN bsolutely RN FFPE Kit RN 10µm2 RN 1 260/ RN 1 bsolutely RN FFPE Kit RN RNng/µl RN ng COPD COPD COPD COPD Stratagene QPCR Reference Total RN, Human Stratagene QPCR Reference Total RN, Human Reference RNPCR Reference RN RN FFPE RN RN Reference RN FFPE RN Reference RN Relative Gene Expression Level in COPD v. Normal NOS1 NOS2 NOS3 COPD enos Nitric oxide synthases (NOS ) L-arginine (L- ) L-citrulline (L- ) (COPD) COPD FFPERN QRT-PCR NOS COPD nnos neuronal nitric oxide synthases inos inducible nitric oxide synthases enos endothelial nitric oxide synthases COPD RN2 3 1 bsolutely RN FFPE Kit QRT-PCRRN 1 COPDeNOS bsolutely RN FFPE Kit # bsolutely RN FFPE Kit ,600 bsolutely RN FFPE Kit ,000 Strategies Vol.2 No.1 p9
11 Cell Biology GFP Problem GFP Solution Vitality hrgfp II EGFP N-C- hrgfp (GFP) GFP N-C- GFP GFP B hrgfp II hrgfp Vitality hrgfp II Protein GIgG1 κ isotype 32kDa EGFP hrgfp GFP Renilla renifornis hrgfp II hrgfp EGFP (equorea Victoria ) 2 ( 1) hrgfp II ( 2) EGFP hrgfp EGFP GFP 1 hrgfp II EGFP HEK-293T 24 : EGFP B: hrgfp II Vitality hrgfp II Vectors and ntibodies Relative Fluorescence Intensity hrgfp II 502 nm 505 nm hrgfp II Wavelength (nm) Vitality hrgfp II # Mammalian Expression Vector Vitality hrgfp II-1 20µg ,000 Vitality hrgfp II-C 20µg ,000 Vitality hrgfp II-N 20µg ,000 Vitality pires-hrgfp II 20µg ,000 Vitality hrgfp II ntibodies hrgfp monoclonal antibody 20µl , µl ,000 Full-length hrgfp polyclonal antibody 20µl , µl ,000 *: Strategies Vol.2 No.1 p10
12 Mutagenesis Problem PCR PCR Solution GeneMorph II Random Mutagenesis Kit PCR GeneMorph II Random Mutagenesis Kit PCR Taq Mutazyme DN TG C 3 4 Mutazyme II DN Taq Mutazyme DN /T C/G 1 Taq PCR MgCl2 dntps PCR PCR % 2 Mut/kb (64 ng) 7.8 (128 ng) 3.8 (1000 ng) 10.4(16 ng) 13 (1.6 ng) (16 pg) duplication # 16pg 1000ng 1kb16 16pg ng GeneMorph II Kit Taq Polymerase Mutazyme II DNTaq PCR0.5mM Mn mM dtp dgtp, 1mM dctp dttp) Taq DN Mutazyme II DN Mutazyme II DN 1. LacZ fragment (650kb) 2. 1kb pbsii clone target 3. Pfu 4. Taq kb pbsii clone target 6. 4kb Pfu /pbsii fragment % Value Mutazyme II DN Polymerase T to N Mutazyme DN Polymerase GC to N Taq2000 DN Polymerase 1 Mutazyme II DN Taq 2~3kb Mutazyme II DN 6kb 3 Random Mutagenesis Kit # GeneMorph II Random Mutagenesis Kit ,000 GeneMorph II EZ Clone Domain Mutagenesis Kit ,000 Strategies Vol.2 No.1 p11
13 Nucleic cid Sample Preparation DN RN Problem PCRcDN Solution SideStep TM Lysis and Stabilization Buffer 10 DN RN cdn SYBR Green PCR DN SideStep TM Lysis and Stabilization Buffer RN SideStep TM Lysis and Stabilization Buffer RN PCRSideStep TM BufferPCRDN PCR1 1 DN RN SideStep TM 1 RN Flourescence (drn) cells 1.75 cells 17.5 cells Cycles SideStep TM QPCR cdn Synthesis Kit700 cdn PCR BX 91.2% Rsq=0.999 BX 700 cdn PCR 1.75 BX 1 RN SideStep TM Buffer RN RN RN SideStep TM Buffer 100 Jurkat T 8gilent Bioanalyzer 28S 18S RN PCR RN20 6 SideStep TM # SideStep TM Lysis and Stabilization Buffer ,400 SideStep TM mrn Enrichment Kit ,000 Brilliant SYBR Green SideStep TM QPCR ,600 Master Mix Strategies Vol.2 No.1 p12
14 FQ SideStep TM SideStep TM Lysis and Stabilization Buffer 1 PCRRT-PCR RN RN SideStep TM PCR bsolutely RN Isolation KitSideStep TM RN Q SideStepTM Buffer RN PCRRT-PCR Q SideStepTM Buffer RN RNRT-PCR bsolutely RN Isolation Kit RN Q SideStepTM SideStep TM RN sirnknock down mirn RN mrn Time course DN RN SideStep TM # SideStep TM Lysis and Stabilization Buffer SideStep TM ,400 SideStep TM mrn Enrichment Kit mrntotal RN ,000 Brilliant SYBR Green SideStep TM QPCR Master Mix SYBR Green DN cdn) ,600 Strategies Vol.2 No.1 p13
15 Topoisomerase I Q SideStepTM BufferRT-PCR 1- step RT-PCR 5,000 1µl 10 4 /µl RNase/DNase 1-step RT-PCR cdn 5,000 7µlPCR 2µl 500 cdncdn Q SideStepTM Buffer SideStep TM laser micro-dissection (LMD) fluorescence-activated cell sorter (FCS) SideStep TM 1 Q SideStepTM RT-PCR RT-PCRDN mrn exon-exon boundary intron DN DN1intron intron cdn 150bp introndn mrn Q 6 RN 6SideStep TM RN gilent bioanalyzer 28S 18S RN RN Integrity Number 8.5 RN 1 SideStep TM mrn Brilliant SideStep TM QRT-PCR Master Mix GPDH Mx3000P QPCRRT-PCR 6-20SideStep TM 1B -80RN 1B Ct SideStep TM mrn 6 [FU] S Marker 28S Time (seconds) B 5 1 Fluorescence (drn) Cells Cycles 50 Cells SideStep TM SideStep TM gilent trace B 6SideStep TM RNRT-PCR Strategies Vol.2 No.1 p14
16 SideStep TM 30%OFF!! # SideStep Lysis and Stabilization Buffer ,800 18,000 SideStep Lysis and Stabilization Buffer ,300 10,700 SideStep mrn Enrichment Kit ,500 67,500 SideStep QPCR cdn Synthesis Kit ,900 51,000 Brilliant SYBR Green SideStep QPCR Master Mix ,600 65,500 Brilliant SYBR Green SideStep QRT-PCR Master Mix, 2-step ,900 99,900 Brilliant SideStep QRT-PCR Master Mix, 1-step ,000 92, StrataCooler 30%OFF!! FullVelocity TM SYBR Green PCR & RT-PCR # FullVelocity SYBR Green QPCR Master Mix ,000 38,400 FullVelocity SYBR Green QRT-PCR Master Mix ,400 61, StrataClone TM 30%OFF!! 2 PCR # StrataClone TM PCR Cloning Kit ,900 41,900 StrataClone TM PCR Cloning Kit ,000 25, jtech@stratagene.com U R L
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