Arrayit Corporation Logo

Arrayit Corporation (ARYC) leads and empowers the genetic, research, pharmaceutical, and diagnostic communities through the discovery, development and manufacture of proprietary life science technologies and consumables for disease prevention, treatment and cure.

Products - Amplification & Labeling - Amino Allyl Labeling Kits For DNA Microarrays

microarray
Arrayit has developed the most robust, comprehensive and complete kits available for amino-allyl cDNA labeling from unamplified or amplified mRNA . The Amino Allyl Labeling Kit is optimized for 1-2 g of mRNA, and is highly recommended for use with our H25K Whole Human Genome Chip. Probe mixtures of cDNAs labeled indirectly using the amino-allyl procedure produce more intense microarray signals and lower background than other labeling procedures. Shown here is a scanned image of H25K hybridized with with 2.0 g of Cy3-labeled cDNA probe derived from total human mRNA and labeled using the Amino Allyl Labeling Kit (Cat. AFK). The intense signal strength and ultra-low background allow users to quantify rare mRNAs not detectable on other platforms and identify biomarkers for major human disease pathways.


Table of Contents

  • Introduction
  • Quality Control
  • Product Advantages
  • Kit Contents
  • Technical Assistance
  • Short Protocol
  • Complete Protocols
  • Recommended adn Required Equipment and Reagents
  • Ordering Information
  • Storage Conditions
  • Warranty

Introduction
Congratulations on taking a big step towards improving the economies of scale, quality and speed of your genomics research. This booklet contains a complete set of protocols outlining the steps and principles needed to use Arrayit Amino Allyl Fluorescent Labeling Kit.

Quality Control
Arrayit assures the performance of this product. The finest scientific research was used to develop this product. Rigorous quality control monitoring of every lot guarantees that these kits conform to the highest industry standards.

Product Advantages

  • Highly recommended for H25K Whole Human Genome Microarrays
  • Supports all substrate and slide surface chemistries
  • Easy protocol, high yields and efficient incorporation
  • Can be used with both two- and three-dimensional surfaces
  • Superior separation chemistry provides 99+% probe purity
  • Dye removal reduces background in microarray hybridizations
  • No alcohol precipitations required
  • Kits arrive ready to use, no buffer or column preparation required
  • Microcentrifuge format allows rapid purification with single columns

Important Note: Spin all sample kit tubes briefly by centrifugation prior to use to ensure proper sample volume.

Kit Contents (20 Labeling Reactions)

Item

Packaging

Storage

Random Primer

44 l in 2 ml tube

-20C Freezer

dTVN Primer  

22 l in 2 ml tube

-20C Freezer

Labeling Enzyme Mix

33 l in 2 ml tube

-20C Freezer

Labeling Reaction Buffer

50 l in 2 ml tube

-20C Freezer

AA-dNTP Mix

90 l in 2 ml tube

-20C Freezer

 DTT

50 l in 2 ml tube

-20C Freezer

cDNA Synthesis Stop Solution

250 l in 2 ml tube

-20C Freezer

Neutralization Solution

250 l in 2 ml tube

-20C Freezer

Dye Binding Buffer

500 l in 2 ml tube

-20C Freezer

Dye Binding Stop Solution

50 l in 2 ml tube

-20C Freezer

Nuclease Free Water

1.5 ml in 2 ml tube

-20C Freezer

Fluorescent Probe Purification Kit

50 reactions per kit

Room temperature


Technical Assistance
Please contact us if you have any comments, suggestions, or if you need technical assistance.  By electronic mail: arrayit@arrayit.com (under the subject heading please type ArrayIt technical assistance).  By telephone: (408) 744-1331, Monday—Friday PST 9:00 am - 4:30 pm. Please remember that we want to hear about your successes!

human_genome_microarray
Figure 1. Scanned single color image of the entire H25K microarray using 2.0 g of Cy3-labeled cDNA probe derived from total human mRNA labeled using the Amino Allyl Labeling Kit (Cat. AFK). Notice the uniformity of the background obtained with probes derived from AFK.


Short Protocol (Steps 1-5)
This kit is optimized for 1-2 g of mRNA as starting material and will generate 0.2-0.5 g labeled cDNA per reaction. The process includes the following steps:

1. cDNA Synthesis from mRNA.
2. Hydrolysis of RNA.
3. Purification of amino allyl labeled cDNA.
4. Dye coupling to amino allyl labeled cDNA.
5. Purification of dye labeled cDNA.

Complete Protocol (Steps 1-5)
1. cDNA Synthesis from mRNA. To a PCR tube on ice add 1-2 g of mRNA or 2-10 g of amplified RNA, 1.0 l of Random Primer, 1.0 l of dTVN Primer, and nuclease free water to 10.5 l total volume. Incubate at 80C for 5 min and allow cooling at room temperature (20-25C) for 10 min. Briefly spin to collect all the liquid and add 1.5 l Labeling Enzyme Mix, 2.0 l Labeling Reaction Buffer, 4.0 l AA-dNTP mix, and 2.0 l DTT. Incubate at 42C for 90 min. Briefly spin to collect all the liquid. Add 10 l of cDNA Synthesis Stop Solution to the mixture and store at -20C.

2. Hydrolysis of RNA. Incubate the cDNA synthesis reaction at 65C for 30 min. Add 10 l of Neutralization Solution to the mixture and mix well.

3. Purification of Aminoallyl Labeled cDNA. To each 100 l amino allyl-labeled cDNA sample, add 300 l of FPP Binding Buffer. Mix thoroughly by vortexing. Transfer the 400 l sample to an FPP Spin Column.  Make sure the Spin Column is seated properly in the 1.5 ml Wash Microfuge Tube. Spin the assembly for 10 sec at 1,000 rpm in a microfuge. Discard the flow-through from Wash Microfuge Tube. Add 125 l FPP Wash Buffer to the Spin Column and spin for 10 sec at 1,000 rpm in a microfuge. Discard the eluent. Wash two more times with 125 l of FPP Wash Buffer. Centrifuge the Spin Column 60 sec in a microfuge to dry completely. Transfer the Spin Column into a sterile Elution Microfuge Tube. Add 50 l of sterile dH20 to the Spin Column and incubate at room temperature for 5 min. Centrifuge the Spin Column for 10 sec at 1,000 rpm to elute the cDNA sample into the Elution Microfuge Tube. Add another 50 l of sterile dH20 to the Spin Column and spin 60 sec at 10,000 rpm to elute any remaining cDNA. Dry the 100 l cDNA sample in a SpeedVac for 30 min at 42C. Store the purified cDNA pellet vacuum-sealed at –80C until the dye coupling step. Store the purified cDNA pellet vacuum-sealed at –80C until the dye coupling step.

4. Dye coupling with Aminoallyl Labeled cDNA. Obtain the dry cDNA samples from -80C. Add 2.5 l of Nuclease Free Water to each cDNA sample. Dissolve the Cy3 or Cy5 mono-reactive dye in 45 l DMSO and mix by vortexing. To each 2.5 l cDNA sample, add 4.5 l of Dye Binding Buffer. To each 7.0 l sample, add 3 l of mono-reactive Cy3 or Cy5 dye for a final reaction volume of 10.0 l. Incubate the 10 l samples at room temperature (20-25C) for 60 min. Quench each 10 l sample by adding 6 l of Dye Binding Stop Solution. Mix well. Incubate at room temperature (20-25C) for 15 min.

5. Purification of Dye Labeled cDNA. Mix both the Cy3 and Cy5 reactions in a single tube. To each 100 l dye -coupled cDNA sample, add 300 l of FPP Binding Buffer. Mix thoroughly by vortexing. Transfer the 400 l sample to an FPP Spin Column.  Make sure the Spin Column is seated properly in the 1.5 ml Wash Microfuge Tube. Spin the assembly for 10 sec at 1,000 rpm in a microfuge. Discard the flow-through from Wash Microfuge Tube. Add 125 l FPP Wash Buffer to the Spin Column and spin for 10 sec at 1,000 rpm in a microfuge. Discard the eluent. Wash two more times with 125 l of FPP Wash Buffer. Centrifuge the Spin Column 60 sec in a microfuge to dry completely. Transfer the Spin Column into a sterile Elution Microfuge Tube. Add 50 l of sterile dH20 to the Spin Column and incubate at room temperature for 5 min. Centrifuge the Spin Column for 10 sec at 1,000 rpm to elute the Cy3-coupled cDNA sample into the Elution Microfuge Tube. Add another 50 l of sterile dH20 to the Spin Column and spin 60 sec at 10,000 rpm to elute any remaining Cy3-coupled cDNA. Dry the 100 l Cy3-coupled cDNA sample in a SpeedVac for 30 min at 42C. Store the purified Cy3-coupled cDNA pellet vacuum-sealed at –80C until the hybridization step. Quantify the cDNA and efficiency of labeling with a low volulme spectrophotometer. Maintain at 4C in the dark until the hybridization step. Alternatively dry the 20 l cDNA samples in a SpeedVac for 30 min at 42C and store the purified cDNA pellet vacuum-sealed at -80C.

Required Equipment and Reagents Not Supplied

  • Ice for incubation
  • Cy3 and Cy5 mono-reactive dye packs (Amersham cat. no. PA23001/ PA25001)
  • PCR tubes
  • Thermal cycler
  • Microfuge capable of 1,000 rpm or more
  • Micropipettors
  • Vortex mixer
  • Vacuum concentrator
  • Low volume Spectrophotometer

Recommended Products

Ordering Information

Product

Description

Catalog ID

Price per kit (US dollars)*

Amino Allyl Fluorescent Labeling Kit

Arrayit Amino Allyl Labeling Kits generate high quality fluorescent cDNA probe mixtures from mRNA to afford intense signals and low background for DNA microarray applications including genomic expression profiling with the H25K Whole Human Genome Microarrays. Kits are optimized for 1-2 g of mRNA as starting material yielding 0.2-0.5 g per reaction, 20 labeling reactions per kit.

AFK

$780Purchase1

*To order Arrayit Products: call 408-744-1331, fax 408-744-1711, email arrayit@arrayit.com or click on the purchase button to proceed directly to the purchase page.

*Add shipping and handling to all orders.

*International pricing may vary as much as 30% (or more depending on country) due to import duties, stocking fees and technical support.

Storage Conditions
Arrayit Amino Allyl Labeling Kits should be stored at –20C. Fluorescent Probe Purification columns and reagents can be stored dry at room temperature (20-25C). The kits are nuclease-free, sterile, and have a shelf life of one-year from the date of purchase.

Warranty
Arrayit Amino Allyl Labeling Kits are sold for research purposes only. Arrayit products are not for use in human diagnostics or for drug purposes, nor are they to be administered to humans in any way.  Extreme care and exact attention should be practiced in the use of the materials described herein.  Arrayit products are subject to extensive quality control and are guaranteed to perform as described when used properly. This product is stable for at least 1 year from the date of purchase when stored at -20C. Any problems with these products should be reported to Arrayit immediately. Our liability is limited to the replacement of the product, or a full refund.  Any misuse of this product is the full responsibility of the user, and Arrayit makes no warranty or guarantee under such circumstances.

Copyright 1993-2014 Arrayit Corporation. All rights reserved.