Overview de soluciones Agilent en Biología

Molecular

Gemma Brils García

Especialista Científico y Comercial de Bioreactivos

April 4, 2014

Confidentiality Label

1

Product categories Homepage

Nucleic Acid Analysis

Reverse Transcription

Amplification

Quantitative PCR

Cell Biology

Cloning

Protein Function and Analysis

Nucleic Acid Analysis

RNA Purification: Absolutely RNA® Purification Kits

RNA: Nanoprep from 200ng, Miniprep > 200μg ofRNA.

Fast extraction from cells and/or tissue, thanks to spin-cups: 30’!

Non toxic: No phenol, no chloroform, no ethanol.

Pre-add DNAse.

Room temperature storage.

Diferentes formatos disponibles, también para extracción de mRNA, miRNA y muestras

parafinadas, ARN plant extraction.

Nucleic Acid Analysis Portfolio

Absolutely RNA®

Miniprep Kit

Absolutely RNA®

Microprep Kit

Absolutely

RNA® 96

Microprep Kit

Absolutely

RNA® Nanoprep

Kit

Absolutely

RNA® FFPE Kit

Absolutely

mRNA™ kit

Sample Size

105 to 107 cells or

5 - 40 mg of tissue Up to 5 x 105 cells

Up to 5 x 105

cells

1 x 104 cells

down to a single

cell

Formalin-fixed

paraffin-

embedded

tissue

Up to 1 mg of

total RNA

Format Spin cup Spin cup Spin cup Spin cup Spin cup

Customized

oligo-dT

magnetic

particles

Capacity > 200 µg RNA 50 µg RNA 50 µg RNA Approx. 200 ng

RNA 50ug

1 mg of total

RNA/ 1mg of

magnetic

particles

Elution

Volume 50 µl 30 µl 30 µl 10 µl 30 µl 10-50 µl

Application

RT-PCR, QRT-

PCR, Northerns,

microarray target

generation, In

vitro transcribed

RNA

Laser

microdissection,

QRT-PCR

microarray target

generation

High throughput

QRT-PCR and

microarray

target

generation

QRT-PCR, LMD QRT-PCR, LMD

RT-PCR, QRT-

PCR, Northerns,

microarray

target

generation

April 4, 2014 Agilent Restricted

4 Stratagene Boot Camp

April 4, 2014

Confidentiality Label

5

DNA: gDNA extraction kit

No tóxico y altamente eficiente: sin fenol ni cloroformo

Desde sangre, tejido y células

También disponible para extracción desde gel de agarosa

Nucleic Acid Analysis

Quantity Yield Size (kb) Time Nº isolations

Sangre

total 5ml >100-300 μg 100-500 2 hours 35 samples at 10ml sample

Tejido 1g >250 50-100 2h 45’ 35 culture pellets at 1x 108 cells/pellet

Cultivo

celular 108 cells >600 50-100 2h 45’ 30 solid samp. 250 mg tissue / samp.

DNA: StrataPrep Plasmid MiniPrep Kit

Accommodates all sizes of DNA from standard 2.9-kb plasmids to 45-kb cosmids

Rapid protocol uses a modified alkaline lysis method, phenol-free

Purified plasmids transform as efficiently as cesium chloride-purified DNA

Reverse Transcription

Característica

principal Nombre Aplicación

Highest Fidelity AccuScript

Cloning

NGS

Protein expression

High sensitivity

High Yield AffinityScript

Multi temperature

RT-PCR

qRT-PCR

Microarray

Gene Expression

High Fidelity

On-tube system

Easy-A One-Tube

Cloning

NGS

Protein expression

AccuScript High Fidelity Reverse Trancriptase

Engineered to deliver

• the highest transcription accuracy possible (3-6 fold fewer errors than conventional RT)

• while promoting full-length cDNA synthesis (up to 20kb) and

• high yield in RT-PCR and superior RT-PCR performance.

April 4, 2014

Confidentiality Label

7

Successful PCR amplification of 0.61kb fragment located at 5’ end of the human nebulin gene, which is 20.8kb in length. This demonstrates complete reverse transcription of the human nebulin gene by AccuScript RT.

Key benefits Product Group

Highest Fidelity PfuUltra II

Superior Yield

Difficult/GC targets Herculase II

Incorporation of dUTP is desired PfuTurbo Cx Polymerase

Sensitivity

Economical PicoMaxx

Long Targets EXL

TA/UA Cloning Easy-A

Most economical Paq5000

Amplification

Pfu Ultra Polymerase

April 4, 2014

Confidentiality Label

9

• Extended length capability : Amplify up to 19 kb

• Contains ArchaeMaxx Factor to prevent poisoning by dUTP

• Optimal PCR cycling at 15 sec/kb; extreme speed leading to reduced overall run time

• Novel hot start technology

• 3-fold higher than any other proofreading enzyme

• 20-fold fewer mistakes than with Taq DNA polymerase

Accuracy of 1: 2.5 x 106 Pfu-based DNA Polymerase Binding Domain

Herculase II polymerase

April 4, 2014

Confidentiality Label

10

• Superior yields and most robust DNA polymerase on the market

• Most economically priced high fidelity enzyme in the market

• Amplification of difficult and GC-rich targets

• Prevent poisoning of dUTP with ArchaeMaxx Factor

• High sensitivity : Amplify from 1 ng of input DNA

• Amplify < 12 kb genomic DNA fast cycling; < 23 kb with optimization

DNA binding domain Pfu-based DNA polymerase

-C Proofreading Polymerization N-

Pfu-based DNA Polymerase Binding Domain

Fidelity of 1/7.5 x 105

April 4, 2014

Confidentiality Label

11

Need help?

Page 12

THERMOCYCLER SURECYCLER 8800

Surecycler software

Page 13

→Easy changeables modular blocks gradient, Fast, 96 and 384 well-plates

→Large touch screen

→Registered programs and 10.000 programs capacity

→Wizard function: make a program in function of your primers sequence or your TM

→96-Well or 384-well user changeable

→One instrument that offers both fast cycling (6°C/sec ramp) and

large volumes capacity (10-100µL)

→Remote access to start, stop, program, and monitor experiments from

most web enabled devices

Quantitative PCR

QPCR & QRT-PCR Reagents: BRILLIANT III

Available:

• Brilliant III Ultra-Fast SYBR® Green

• Brilliant III Ultra-Fast probe based

Starting material:

• From RNA: qRT-PCR

• From DNA: qPCR

Genes:

• No-Multiplex: 2 genes

• Multiplex: up to 5 genes

Light emission

Light

energy transfer

Taq

unbound probe free in solution

Reporterdye

Acceptordye (Quencher)

Taq

Light

Rox: Reference Passive Dye

• High Concentration of ROX

• Low Concentration of ROX

• Separate ROX

ROX PRODUCT COMPATIBILITY

Brilliant III QPCR

Low ROX Master Mix

MX 3000p, Mx 3005p

7500 and 7500 FAST Real-Time PCR

System

Viia7 Real-Time PCR System

Brilliant III QPCR

High ROX Master Mix

7900 y 7900HT Real-Time PCR System

StepOne y StepOne Plus Real-Time CR

System

Roche LC 480

Eppendorf

Brilliant III QPCR

Separate ROXMaster Mix

CXF96 BioRad

QIAGEN Rotor-Gene Q

2x MM

+ ROX

Sample

No ROX normalization With ROX normalization

QPCR & QRT-PCR Reagents:

SideStep II QRT-PCR

Lysis & stabilization products

One-step QRT-PCR quantification of RNA directly from cells

without RNA purification (probe-based detection)

Save money: Purify only relevant samples after screening

Archive samples: RNA stabilized up to 6 months

Save time: High throughput amenable

Accurate data: RNA loss minimized

Safe: No hazardous waste disposal and removal

April 4, 2014

Confidentiality Label

16

High-Specificity miRNA QRT-PCR Detection Kit

QPCR Reference Total RNA

Alien® QRT-PCR Inhibitor Alert

Page 17

Real-time Thermocycler Mx 3000p and 3005p

→The Largest range of excitation

(350nm - 750nm)

→Photo-Multiplicator Tube (PMT) in

detection for a better sensitivity

→Open system (use consumables and

reagents with no constraint)

→Software (MxPro) Easy to use

Cell Biology

QPCR and PCR Based Mycoplasma Detection

• QPCR and PCR Assay Kits

• 8 most common cell culture Mycoplasma contaminants, including the often illusive Acholeplasma

laidlawii.

PRD000164_MycoSensor™ QPCR Assay Kit

Cloning

XL10-Gold Ultracompetent Cells

StrataClone™ PCR Cloning Kits

- PCR Based: User can choose the enzyme (PfuUltra & Herculase II

recommended)

- FAST: 5 minute ligation into vector arms

- Transform and plate

- Colony screening and confirmation

- Transfect clone of interest into mammalian cell for protein expression

- 5 varieties of vector arms are available, with either an N- or C-terminal tag

(FLAG or c-Myc) or no tag

StrataClone Product(s)

StrataClone PCR Cloning Kit

StrataClone Blunt PCR Cloning Kit

StrataClone Mammalian Expression Vector Systems

Protein Expression: Competent Cell Portfolio

• 5x109 transformation efficiency of large DNA molecules

• Permits transformation of methylated products

XL10-Gold Ultracompetent Cells

• Stop Unwanted Rearrangement Events

• Successfully clone unstable DNA SURE

• Clone DNA encoding toxic proteins

• Reduce plasmid copy number to increase cell viability

ABLE

• Overcomes codon bias problems

• Increases supply of rare E.coli tRNAs BL21 CodonPlus

• Low growth temperatures enhance protein folding and solubility

• Correct for codon bias and enhance solubility at the same time

ArcticExpress

Protein Function and Analysis

DIRECTED:

Quik Change Lightning Site Directed Mutagenesis kit

Quik Change Lightning Site Directed Mutagenesis kit

• Mutagénesis (cambios de base), Inserciones, Delecciones,..

RANDOM:

GeneMorph II Random Mutagenesis

Protein Expression: Transfection and Gene Delivery

DNA Transfection:

- GeneJammer®: polyamine formulation

- LipoTAXI: liposome-mediated transfection

- Mammalian Transfection Kit: CaPO4 and DEAE

Viral Mediated Gene Delivery:

- ViraPack™ Transfection Kit: Integrates into host chromosome

THANK YOU