Optimice sus purificaciones de ADN y ARN con nuestros kits Stratagene y el Bioanalizador 2100
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
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
INTRODUCCIÓN; Extracción de ADN
DNA Extraction Ref 200600
Stratagene s Products for DNA Purification StrataPrep Plasmid Miniprep Kit (ref 400761) StrataPrep Gel Extraction Kit (ref 400766) StrataPrep PCR Purification Kit (ref 400771) Lambda DNA Purification Kit (ref 200391)...Rapid Lambda miniprep method NucTrap Probe Purification Columns ref (400701)...Purifies>17bp DNA or RNA fragments in two minutes.
Strataprep Plasmid Miniprep kit (ref 400761) -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
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
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
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 (25 12000 bp) Integrity check, Separation and Quantitation of RNAs Cell Fluorescence Assays with stained cells (Apoptosis, Transfection, Expression, ) Page 10 Agilent 15 Marzo 2010
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
0. preparatory transformations Noise reduction (Savitzky-Golay filtering Spike rejection Baseline correction Baseline correction disabled Baseline correction enabled Agilent 15 Marzo 2010 Page 12
1. Peak recognition table of fluorescence intensities table of peaks Electropherogram in Expert Result table in Expert Agilent 15 Marzo 2010 Page 13
2. Alignment to markers Upper marker Lower marker Analysis turned off Unaligned data Analysis turned on Aligned data Agilent 15 Marzo 2010 Page 14
3. Sizing size: ladder Agilent 15 Marzo 2010 Page 15
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Detección de SNPs en los genes p16 y K-ras Page 17
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
Aplicaciones con DNA Marcos Morey Hospital Clínico de Santiago de Compostela. marcos.morey@usc.es
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
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
RIN Integridad del ARN Page 22
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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
Absolutely mrna Purification Kit (ref 400806) 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
Análisis del mrna Contaminación con rrna de muestras de mrna Page 26
Absolutely RNA Microprep Kit (ref 400805) 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
Microdisección y análisis de ARN Resultado de la microdisección 1.000 células (130-700 pg/ul) Suficiente para la optimización de una técnica? RNA 6000 pico Page 28
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
Absolutely RNA FFPE kit (ref 400809) 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
Absolutely RNA Miniprep Kit (ref 400800) 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
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
Absolutely RNA Isolation Kits
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
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
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
Absolutely RNA mirna kit (ref 400814) -Selecting the Appropriate Protocol -Optimal Sample Sizes and Yield Guidelines -Sample Viscosity Considerations -RNA and RNase Levels in Different Tissues
Small RNA kit Page 38
Small RNA distinción de especies de small RNA total) Análisis de RNAs de células Schneider de Drosophila (10ng RNA Page 39
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
SideStep kits
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
Gracias a todos!!! Hugo_mingo@agilent.com Page 43