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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
• 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
sample prep > amplification > cloning
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
3/6/2010 Page 8
From Cell to Gel
Cells
Proteins DNA
LC, ESI-MS, MALDI Bio Assays
…
SDS-PAGE
MALDI Hybridizations
…
Agarose-Gels
PCR
Amplification DNA
Page 9
2100 Bioanalyzer Hardware
Exchangeable cartridge
for electrophoresis or flow
cytometry assays
16 pin electrodes
connected to HV-sources
Chip holder with
heater plate
Optics for detection
Chip
selector
Agilent
Agilent 15 Marzo 2010
Page 10
The Agilent 2100 bioanalyzer offers
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, )
Improved quality of slab-gel-type analysis
through on-Chip electrophoresis…
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
…reducing the experiment to:
Agilent 15 Marzo 2010 Page 11
Lab-on-a-Chip - Principle of Injection & Separation
3
2
4
Direction of electrodriven movement of liquids and molecules within liquids
1
Separation channel Inje
ction
ch
an
ne
l
Agilent 15 Marzo 2010
Page 12
0. preparatory transformations
• Noise reduction
(Savitzky-Golay filtering
• Baseline correction
• Spike rejection
Baseline correction disabled Baseline correction enabled
Agilent 15 Marzo 2010
Page 13
1. Peak recognition
table of
fluorescence
intensities
table of
peaks
Result table in Expert
Electropherogram in Expert
Agilent 15 Marzo 2010
Page 14
2. Alignment to markers
Lower marker
Upper marker
Analysis turned off –
Unaligned data
Analysis turned on –
Aligned data
Agilent 15 Marzo 2010
Page 15
3. Sizing
size:
ladder
Page 16
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.
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
Page 23
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 (5x105 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
Page 28
Resultado de la microdisección 1.000 células (130-700 pg/ul)
¿Suficiente para la optimización de una técnica?
RNA 6000 pico
Microdisección y análisis de ARN: contaminación
por ADN de la muestra (riñon de ratón)
Page 29
Sobreestimación
de la concentración
Efectos de la digestión de ADN en
columna con DNasa
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 105 to 107 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 (<1x104 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
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
Análisis de RNAs de células Schneider de Drosophila (10ng RNA
total)
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
