Indirect Amino Allyl Fluorescent Labeling Kit

Products - Amplify & Label - Amino Allyl Labeling Kits

This kit is optimized to use with 1-2 µg of mRNA as starting material. The process includes the following steps:

cDNA Synthesis from mRNA.
Hydrolysis of RNA.
Purification of Amino Allyl labeled cDNA.
Dye binding with Amino Allyl labeled cDNA.
Purification of dye labeled cDNA. When used properly this kit will generate 200-500ng of dye labeled cDNA.

Can be used stand alone or in conjunction with:

Table of Contents

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^® Indirect Amino Allyl Fluorescent Labeling Kit.

Quality Control
TeleChem assures the performance of this product. The finest scientific research went into the development of this product. Rigorous quality control monitoring on a lot-by-lot basis guarantees that the ingredients conform to the highest industry standards.

Product Advantages

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

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 9:00am - 4:30pm. Please remember that we want to hear about your successes!

Kit Contents

Item

Packaging

Storage

Random Primer

22 µl in 0.5ml tube

-20^oC Freezer

dTVN Primer

22 µl in 0.5ml tube

-20^oC Freezer

Labeling Enzyme Mix

33 µl in 0.5ml tube

-20^oC Freezer

Labeling Reaction Buffer

44 µl in 0.5ml tube

-20^oC Freezer

AA-dNTP Mix

88 µl in 0.5ml tube

-20^oC Freezer

DTT

22 µl in 0.5ml tube

-20^oC Freezer

cDNA Synthesis Stop Solution

220 µl in 0.5ml tube

-20^oC Freezer

Denaturing Solution

220 µl in 0.5ml tube

-20^oC Freezer

Neutralization Solution

220 µl in 0.5ml tube

-20^oC Freezer

Dye Binding Buffer

500 µl in 2ml tube

-20^oC Freezer

Dye Binding Stop Solution

110 µl in 0.5ml tube

-20^oC Freezer

DNA Binding Buffer

6.6 ml in 8ml bottle

Room Temp

DNA Wash Buffer Concentrate

1.5 ml in 8ml bottle

Room Temp

DNA Micro Column

30

Room Temp

Elution Tube (1.5ml)

30

Room Temp

2 ml Wash Tube

30

Room Temp

Nuclease Free Water

1.5ml in 2ml tube

-20^oC Freezer

Requirements & Recommendations

Universal Reference mRNA for Human, Mouse and Rat
Micro-Total RNA Extraction Kit
MiniAmp mRNA Amplification Kit

Items needed but not supplied:

Reactive Dye such as Amersham CyDye™ Set Cat # RPN5661

Alexa Fluor® 555 and Alexa Fluor® 647 reactive dyes
Pierce DyLight 547 and 647 Fluors or other mono reactive dye suitable for microarray applications.

Ethanol 100%
Ethanol 80%
Ice for incubation
PCR tubes

Equipment needed

Thermal cycler
Microfuge capable of 10,000 rpm or more
Micropipettors
Vortex mixer

Recommended
NanoDrop ND-1000A Spectrophotometer. These units work with 1 µl samples and eliminate the need for dilutions, cuvettes, and capillaries. This innovative product is capable of measuring extremely low amounts of nucleic acid and outperforms traditional spectrophotometers at high concentrations.

Short Protocol
This kit is optimized to use with 1-2 µg of mRNA as starting material. The process includes the following steps:

cDNA Synthesis from mRNA
Hydrolysis of RNA
Purification of Amino allyl labeled cDNA
Dye binding with Amino allyl labeled cDNA
Purification of dye labeled cDNA

If used properly this kit will generate 200-500ng of dye labeled cDNA for hybridization with microarray

IMPORTANT Note: Spin all tubes briefly before opening.

Complete Protocol
Step 1. cDNA Synthesis from mRNA.
In a PCR tube on ice add the following:

1-2 µg of mRNA

x µl

Random Primer

1 µl

dTVN Primer

1 µl

Nuclease free water to

10.5 µl

Incubate at 70^oC for 5 mins and allow cooling at RT for 10 mins for primer annealing.

Briefly spin to collect all the liquid

Labeling Enzyme Mix

1.5 µl

Labeling Reaction Buffer

2 µl

AA-dNTP mix

4 µl

DTT

1 µl

Incubate at 42^oC for 90 mins.
Briefly spin to collect all the liquid.
Add 10 µl of cDNA Synthesis Stop Solution to the mixture.
Store at -20^oC.

Step 2. Hydrolysis of RNA.

Add 10 µl of Denaturing Solution to the mixture
Incubate at 65^oC for 30 mins
Add 10 µl of Neutralization Solution to the mixture

Step 3. Purification of Amino Allyl Labeled cDNA.
Before Starting add 6ml of 100% ethanol to DNA Wash Buffer Concentrate.
Add 200 µl of DNA binding buffer to the mixture and load onto a Micro DNA Column.

Load the entire contents onto a DNA Micro Column and place the column in a 2 ml wash tube
Centrifuge for 1 min at full speed in a microfuge (>10000 rpm), discard flow through and reuse wash tube
Add 100 µl of DNA wash buffer to the column and spin at full speed for 1 min
Add another 100 µl of DNA wash buffer to the column and spin at full speed for 2 min
Note: Longer centrifugation time may be necessary to make the column free of ethanol
Discard the 2 ml wash tube and carefully place the column in an elution tube
Add 10 µl of Dye Binding Buffer that has been pre-warmed to 60^oC to the center of the column
Wait for 2 mins and spin at full speed for 1 min
Reload the filtrate on the column
Wait for 2 mins and spin at full speed for 1 min

Discard the column and use the eluted Amino Allyl cDNA in the next step.
Store at -20^oC.

Step 4. Dye Binding with Amino allyl Labeled cDNA.

Add the entire amount (8-10 µl) of eluted cDNA into one Cy dye NHS ester tube (Amersham Cat # RPN5661) and re-suspend briefly using a pipette tip.
Add 8 µl Dye Binding Buffer Incubate at Room Temperature in Dark with shaking at about 60 RPM for 120 mins.
Add 5 µl Dye Binding Stop Solution to stop the reaction. Incubate at Room Temperature in Dark with shaking at about 60 RPM for 30 mins. Mix both the reactions in a single tube.

Step 5. Purification of Dye Labeled cDNA.
Add 200 µl of DNA binding buffer to the mixture and load onto a Micro DNA Column.

Load the entire contents onto a DNA Micro Column and place the column in a 2 ml wash tube
Centrifuge for 1 min at full speed in a microfuge (>10000 rpm), discard flow through and reuse wash tube
Add 100 µl of DNA wash buffer to the column and spin at full speed for 1 min
Add another 100 µl of DNA wash buffer to the column and spin at full speed for 2 min
Note: Longer centrifugation time may be necessary to make the column free of ethanol
Discard the 2 ml wash tube and carefully place the column in an elution tube
Add 10 µl of nuclease free water that has been pre-warmed to 60^oC to the center of the column
Wait for 2 mins and spin at full speed for 1 min
Add 10 µl of nuclease free water that has been pre-warmed to 60^oC to the center of the column
Wait for 2 mins and spin at full speed for 1 min

Discard the column and use the eluted labeled cDNA for hybridization.
Maintain at 4^oC until pre-hybridization.

Troubleshooting Tips

Ordering Information

Product

Description

Catalog ID

Price per kit (US dollars)*

ArrayIt Indirect Amino Allyl Fluorescent Labeling Kit

This kit is optimized to use with 1-2 µg of mRNA as starting material. The process includes the following steps: (1) cDNA Synthesis from mRNA (2) Hydrolysis of RNA (3) Purification of Amino allyl labeled cDNA (4) Dye binding with Amino allyl labeled cDNA (5) Purification of dye labeled cDNA. When used properly this kit will generate 200-500 ng of dye labeled cDNA for hybridization. Twenty (20) Labeling Reactions for ten (10) Two Color Microarrays.

AFK

$550.00

*To order ArrayIt^® Brand Products: call 408-744-1331, fax 408-744-1711, email arrayit@arrayit.com or click on the button below to go directly to the purchase page.

*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® Red and Green should be stored at –20 °C. Fluorescent Probe Purification columns and reagents can be stored dry at room temperature (20-25°C). The kits perform well across a wide range of ambient temperatures and relative humidity. The kits are nuclease-free, sterile, and have a shelf life of one-year from the date of purchase.

Warranty
ArrayIt® brand products have been scientifically developed and are sold for research purposes. Extreme care and exact attention should be practiced in the use of the materials described herein. All ArrayIt® brand products are subject to extensive quality control and are guaranteed to perform as described when used properly. Any problems with our ArrayIt® brand product should be reported to TeleChem immediately. TeleChem’s liability is limited to the replacement of the product, or a full refund. Any misuse of this product including deviations from our protocols is the full responsibility of the user, and TeleChem makes no warranty or guarantee under such circumstances.