Optimice sus purificaciones de ADN y ARN con nuestros kits Stratagene y el Bioanalizador 2100

Transcription

1 Optimice sus purificaciones de ADN y ARN con nuestros kits Stratagene y el Bioanalizador 2100

2 Indice 1-Introducción a la purificación del ADN A- kits de extracción B- Control de Calidad 2-Introducción a la purificación de ARNt y ARNm A- kits de extracción B- Control de Calidad 3-Extracción de mi RNA

3 Products portfolio Agilent sample prep > amplification > cloning Sample Prep Purified Human RNAs DNA Isolation Kits Absolutely RNA purification kits PCR Easy-A DNA polymerase PfuUltra II DNA polymerase PfuTurbo DNA polymerase Herculase II DNA polymerase EXL DNA polymerase RT-PCR AffinityScript RT AccuScript RT-PCR kits cdna Synthesis Kit Competent Cells XL10-Gold ultracompetent cells ElectroTen-Blue electroporation competent cells XL1-Blue series Vectors StrataClone PCR Cloning Kits

4 INTRODUCCIÓN; Extracción de ADN

5 DNA Extraction Ref

6 Stratagene s Products for DNA Purification StrataPrep Plasmid Miniprep Kit (ref ) StrataPrep Gel Extraction Kit (ref ) StrataPrep PCR Purification Kit (ref ) Lambda DNA Purification Kit (ref )...Rapid Lambda miniprep method NucTrap Probe Purification Columns ref (400701)...Purifies>17bp DNA or RNA fragments in two minutes.

7 Strataprep Plasmid Miniprep kit (ref ) -For high yields of plasmid DNA - up to 20 µg from 1.5 to 3.0 ml of culture Superior purity - DNA transforms E. coli with high efficiency and yields quality sequence using automated or manual methods -Accommodates all sizes of DNA from standard 2.9-kb plasmids to 45-kb cosmids -Purified plasmids transform as efficiently as cesium chloride-purified DNA -Rapid protocol uses a modified alkaline lysis method

8 From Cell to Gel DNA Proteins Cells LC, ESI-MS, MALDI Bio Assays SDS-PAGE DNA PCR Amplification MALDI Hybridizations Agarose-Gels Page 8 3/6/2010

9 2100 Bioanalyzer Hardware Exchangeable cartridge for electrophoresis or flow cytometry assays 16 pin electrodes connected to HV-sources Chip holder with heater plate Chip selector Optics for detection Agilent Page 9

10 The Agilent 2100 bioanalyzer offers Improved quality of slab-gel-type analysis through on-chip electrophoresis reducing the experiment to: Easy to use analytical device that performs separation and quantitation of RNA, DNA and Proteins based on electrophoresis within a disposable glass chip. 1. Load sample 2. Run analysis 3. Analyze data On a single Platform perform: Sizing, Quantitation and Purity of Proteins (5-230 kda) Sizing, Quantitation and Purity of DNA fragments ( bp) Integrity check, Separation and Quantitation of RNAs Cell Fluorescence Assays with stained cells (Apoptosis, Transfection, Expression, ) Page 10 Agilent 15 Marzo 2010

11 Injection channel Lab-on-a-Chip - Principle of Injection & Separation 1 2 Separation channel 3 4 Direction of electrodriven movement of liquids and molecules within liquids Page 11 Agilent 15 Marzo 2010

12 0. preparatory transformations Noise reduction (Savitzky-Golay filtering Spike rejection Baseline correction Baseline correction disabled Baseline correction enabled Agilent 15 Marzo 2010 Page 12

13 1. Peak recognition table of fluorescence intensities table of peaks Electropherogram in Expert Result table in Expert Agilent 15 Marzo 2010 Page 13

14 2. Alignment to markers Upper marker Lower marker Analysis turned off Unaligned data Analysis turned on Aligned data Agilent 15 Marzo 2010 Page 14

15 3. Sizing size: ladder Agilent 15 Marzo 2010 Page 15

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17 Detección de SNPs en los genes p16 y K-ras Page 17

18 Inestabilidad de microsatélites en carcinoma sin marcaje adicional Comparación entre tejido normal y tumoral para el análisis del patrón de los microsatélites tras amplificación por PCR. Page 18

19 Aplicaciones con DNA Marcos Morey Hospital Clínico de Santiago de Compostela.

20 Integridad del ARN Calidad del ARN total de la muestra Paso clave en trabajo tras la preparación de la muestra de ARN anterior a realtime- PCR o microarrays ARN de alta calidad ARN de baja calidad Page 20

21 RIN RNA Integrity Number The ratio of ribosomal bands is not sufficient to describe RNA integrity! RNA degradation is a gradual process. Results have to be interpreted other than by visual inspection. Overlay of electropherograms only works well for samples with the same concentration. Instrument dependency in signal height Page 21

22 RIN Integridad del ARN Page 22

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24 Traditional Method for RNA Isolation Guanidine, Phenol, Chloroform method Not effective with small samples Requires precipitation of RNA (yield loss with small samples) Limited application (i.e., no laser capture microdissection) Organic waste disposal Phenol contamination problems No DNA removal step

25 Absolutely mrna Purification Kit (ref ) Precision engineered magnetic bead technology delivers the purest mrna possible Specially constructed beads have a large surface area to maximize yields of polya-rna Unique Outer Shell Prevents Magnetite Recovery Optimized buffer system prevents non-specific binding of rrna and minimizes rrna contamination Fast 20 minute protocol Scalable, simple kit method

26 Análisis del mrna Contaminación con rrna de muestras de mrna Page 26

27 Absolutely RNA Microprep Kit (ref ) Laser Capture Microdissection (LCM) cells Works with as little as 1 cell (5x10 5 cells) Elution 30ul Perfect kit for when starting materials are scarce On column DNase digestion No phenol extraction or ethanol ppt Great for RT-PCR Fast and easy protocol Store at RT

28 Microdisección y análisis de ARN Resultado de la microdisección células ( pg/ul) Suficiente para la optimización de una técnica? RNA 6000 pico Page 28

29 Microdisección y análisis de ARN: contaminación por ADN de la muestra (riñon de ratón) Efectos de la digestión de ADN en columna con DNasa Sobreestimación de la concentración Page 29

30 Absolutely RNA FFPE kit (ref ) A simple, reliable method for isolation of quality RNA from paraffin-embedded tissue sections On-column RNA separation Phenol-free Each lot tested by real-time QPCR Human reference RNA provided in kit Effective on human and mouse tissues

31 Absolutely RNA Miniprep Kit (ref ) Designed specifically for RT-PCR with highly stringent quality controls conventional RT-PCR real time quantitative RT-PCR Fast and easy protocol Room Temperature storage DNase digestion performed on column (no phenol extraction or ethanol ppt) Purifies total RNA from cells and tissue Ideal for samples 10 5 to 10 7 cells or 5 to 40mg of tissue

32 Absolutely RNA Nanoprep Kit Perfect kit for when starting materials are scarce and when concentrated samples are needed Works with very limited samples (<1x10 4 cells) On column DNase digestion No phenol extraction or ethanol ppt Great for RT-PCR Fast and easy protocol Store at RT Elution 10ul

33 Absolutely RNA Isolation Kits

34 Extracción de ARNt y Cuantificación Raul M. Luque, PhD "Ramon y Cajal" Researcher University of Cordoba, Dpt. of Cell Biology, Physiology and Immunology, Section of Cell Biology bc2luhur@uco.es

35 What Are micrornas (mirnas)? Small regulatory hairpin shaped RNAs Bind to mrna to interrupt transcription Implicated heavily in Disease Development Cancer 250 mirnas identified, increasing weekly

36 Absolutely RNA mirna kit ref Latest addition to the nucleic acid isolation kit portfolio produce high yields of pure mirna containing total RNA from cells or tissue samples Compatible with most cell and tissue types specifically qualified for RT-PCR, QRT-PCR and microarray applications Fast and Simple 30-minute procedure with no tedious ethanol precipitations

37 Absolutely RNA mirna kit (ref ) -Selecting the Appropriate Protocol -Optimal Sample Sizes and Yield Guidelines -Sample Viscosity Considerations -RNA and RNase Levels in Different Tissues

38 Small RNA kit Page 38

39 Small RNA distinción de especies de small RNA total) Análisis de RNAs de células Schneider de Drosophila (10ng RNA Page 39

40 SideStep Easy to use lysis buffer for tissue and cultured cells one step protocol, lyses cells and stabilizes nucleic acids RNA stabilization at least 20 months (-80 C) or 6 month at -20 C prevents loss or degradation SideStep lysate makes RNA and DNA available for amplification Total RNA and mrna can be isolated from SideStep lysate Isolated Nucleic Acids can be used for QPCR directly

41 SideStep kits

42 Promoción válida asistentes 40% de descuento en cualquier kit de purificación de ADN o ARN. 25% de descuento en chips para Bioanalizador ADN y ARN

43 Gracias a todos!!! Hugo_mingo@agilent.com Page 43