Reagents - Microarray Buffers - Microarray Wash Buffers to Enhance VIP, Gene Expression, SNP Genotyping and CGH DNA Microarray Assays

Arrayit announces an improved and expanded product line of microarray buffers for nucleic acid microarrays. Wash Buffers A, B and C are used for cDNA and long oligonucleotide microarrays and Wash Buffers 1, 2 and 3 are designed for short oligonucleotide microarrays. Each is composition-optimized to provide the greatest hybridization stringency possible, while preserving intense signals and ensuring low background.  Our wash buffers are supplied as 10X concentrates in one-liter volumes and the ultra-pure reagents are subjected to 0.2-µm filtration and extensive quality control and quality assurance for optimal performance. Arrayit Wash Buffers remove the guesswork from the post-hybridization microarray washing process.

Table of Contents

• Introduction
• Quality Control
• Product Description
• Technical Assistance
• Short Protocol (Wash Buffers A-C)
• Complete Protocol (Wash Buffers A-C)
• Short Protocol (Wash Buffers 1-3)
• Complete Protocol (Wash Buffers 1-3)
• Troubleshooting Tips
• Recommended Products
• Ordering Information
• Warranty

Introduction
The use of Arrayit brand Wash Buffers will improve the precision, speed and affordability of your microarray research in genomics, biomedicine, pharmaceuticals, diagnostics and agriculture. This handbook contains all the information required to take full advantage of Arrayit's Arrayit brand Microarray Wash Buffers.

Quality Control
Arrayit uses the highest quality control (QC) and quality assurance (QA) measures to ensure the quality of our Arrayit brand Microarray Wash Buffers. The finest science and engineering was used to develop this product line. The use of advanced R&D, ultra-pure reagents, and 0.2 µm filtration guarantees that this product line outperforms the highest industry standards.

Product Description
Arrayit brand Microarray Wash Buffers A, B & C and 1, 2 & 3 are designed for post-hybridization washing of cDNA and long oligonucleotide microarrays (Wash Buffers A, B & C) and short oligonucleotide microarrays (Wash Buffers 1, 2 & 3). Users will appreciate the following features:

• Based on the finest microarray research and development (R&D)
• Excellent for gene expression and genotyping applications
• Designed for cDNAs and long oligos (A, B & C) and short oligos (1, 2 & 3)
• High stringency without reducing signal intensities
• Superior formulation reduces background
• Subjected to lot-by-lot quality control (QC) and quality assurance (QA)
• Ultra-pure reagents used for buffer formulation
• All buffers purified by 0.2 µm sterile filtration
• Provided in 1 liter bottles as 10X concentrates
• Each liter of 10X buffer will wash up to 500 microarrays
• Arrive pre-mixed and ready to use
• Remove the guesswork from post-hybridization wash step

Technical Assistance
Please contact us if you have any questions, comments, suggestions, or if you need technical assistance.  By electronic mail, use arrayit@arrayit.com and type "ArrayIt technical assistance" into the subject line. By email, arrayit@arrayit.com between the hours of 8AM and 7PM PST Monday through Friday.  We want to hear about your successes and are always happy to publish contributed sample data on our website.

Short Protocol for cDNAs and long oligonucleotides (Wash Buffers A-C)
1. Remove cDNA or long oligonucleotide microarray from hybridization cassette.
2. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer A.
3. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer B.
4. Dunk for 1 sec at room temperature in 50 ml 1X Wash Buffer C.
5. Spin dry and scan.

Complete Protocol for cDNAs and long oligonucleotides (Wash Buffers A-C)
1. Remove cDNA or long oligonucleotide microarray from hybridization cassette. Following the 1-4 hour hybridization reaction, remove the microarray from the hybridization cassette and proceed quickly to Step 2 to prevent cooling or drying of the microarray.  Arrayit  provides Hybridization Cassettes in a variety of designs and colors, and our cassettes are recommended for best experimental results.

2. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer A.  To a High-Throughput Wash Station, add 50 ml of 10X Wash Buffer A and 450 ml distilled H2O to make 500 ml of 1X Wash Buffer A. Mix the 500 ml of buffer for 30 seconds before adding the microarrays. A Wash Station containing 500 ml of 1X Wash Buffer A is sufficient to wash 1- 25 glass microarrays (25 x 76 mm). Wash the microarrays for 5 minutes at room temperature using moderate to vigorous agitation.  After approximately 30 seconds, the cover slips will loosen from the surface and should be removed carefully with a forceps to prevent the cover slips from scratching the microarrays during the wash process. Wash Buffer A is a low stringency wash buffer designed to remove excess fluorescent probe material from the surface of cDNA and long oligonucleotide (>25-mer) microarrays.  After 5 minutes in 1X Wash Buffer A, proceed quickly to the next step.

3. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer B.  To a second High-Throughput Wash Station, add 50 ml of 10X Wash Buffer B and 450 ml distilled H2O to make 500 ml of 1X Wash Buffer B. Mix the 500 ml of buffer for 30 seconds before adding the microarrays. A Wash Station containing 500 ml of 1X Wash Buffer B is sufficient to wash 1- 25 glass microarrays (25 x 76 mm). Transfer the black substrate rack containing the microarrays from Wash Buffer A to Wash Buffer B. The transfer should be made quickly to prevent drying. Wash the microarrays for 5 minutes at room temperature in Wash Buffer B using moderate to vigorous agitation.  Wash Buffer B is a high stringency wash buffer designed to remove excess fluorescent probe material from the surface of cDNA and long oligonucleotide (>25-mer) microarrays. Additional stringency can be imparted by elevating the temperature of Wash Buffer B to either 37°C or 42°C. Buffer should be pre-heated in a microwave oven to the desired temperature before adding it to the Wash Station.  After 5 minutes in 1X Wash Buffer B (25-42°C), proceed quickly to the next step.

4. Dunk for 1 sec at room temperature in 50 ml 1X Wash Buffer C. To a 50 ml conical tube, add 5 ml 10X Wash Buffer C and 45 ml distilled H2O. Mix thoroughly by vortexing before adding the microarrays. Transfer microarrays one at a time from Wash Buffer B to the 50 ml tube containing Wash Buffer C. Dunk each microarray for 1 second to remove Wash Buffer B and proceed quickly to the next step. Wash Buffer C is designed to remove Wash Buffer B components that will cause streaks on the surface if not removed.

5. Spin dry and scan. Transfer the microarray quickly from Wash Buffer C to a Microarray High-Speed Microarray Centrifuge and spin at full speed for 10 seconds to dry the microarray.  The microarray surface should be clean, dry and ready for scanning at this stage.

Short Protocol for short oligonucleotides (<25-mer) (Wash Buffers 1-3)
1. Remove short oligonucleotide (<25-mer) microarray from hybridization cassette.
2. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer 1.
3. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer 2.
4. Dunk for 1 sec at room temperature in 50 ml 1X Wash Buffer 3.
5. Spin dry and scan.

Complete Protocol for short oligonucleotides (<25-mer) (Wash Buffers 1-3)
1. Remove short oligonucleotide (<25-mer) microarray from hybridization cassette.  Following the 1-4 hour hybridization reaction, remove the microarray from the hybridization cassette and proceed quickly to Step 2 to prevent cooling or drying of the microarray. Arrayit provides Hybridization Cassettes in a variety of designs and colors, and our cassettes are recommended for best experimental results.

2. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer 1.  To a High-Throughput Wash Station, add 50 ml of 10X Wash Buffer 1 and 450 ml distilled H2O to make 500 ml of 1X Wash Buffer 1.  Mix the 500 ml of buffer for 30 seconds before adding the microarrays.  A Wash Station containing 500 ml of 1X Wash Buffer 1 is sufficient to wash 1- 25 glass microarrays (25 x 76 mm).  Wash the microarrays for 5 minutes at room temperature using moderate to vigorous agitation.  After approximately 30 seconds, the cover slips will loosen from the surface and should be removed carefully with a forceps to prevent the cover slips from scratching the microarrays during the wash process. Wash Buffer 1 is a low stringency wash buffer designed to remove excess fluorescent probe material from the surface of short oligonucleotide (<25-mer) microarrays.  After 5 minutes in 1X Wash Buffer 1, proceed quickly to the next step.

3. Wash for 5 min at room temperature in 500 ml 1X Wash Buffer 2.  To a second High-Throughput Wash Station, add 50 ml of 10X Wash Buffer 2 and 450 ml distilled H2O to make 500 ml of 1X Wash Buffer 2. Mix the 500 ml of buffer for 30 seconds before adding the microarrays. A Wash Station containing 500 ml of 1X Wash Buffer 2 is sufficient to wash 1- 25 glass microarrays (25 x 76 mm). Transfer the black substrate rack containing the microarrays from Wash Buffer 1 to Wash Buffer 2. The transfer should be made quickly to prevent drying. Wash the microarrays for 5 minutes at room temperature in Wash Buffer 2 using moderate to vigorous agitation.  Wash Buffer 2 is a high stringency wash buffer designed to remove excess fluorescent probe material from the surface of short oligonucleotide (<25-mer) microarrays. Additional stringency can be imparted by elevating the temperature of Wash Buffer 2 to either 37°C or 42°C. Alternatively, Wash Buffers 1 and 2 can be cooled from 4-20°C to reduce the stringency.  Buffers should be pre-heated or cooled to the desired temperature and then transferred to the Wash Station. After 5 minutes in 1X Wash Buffer 2 (4-42°C), proceed quickly to the next step.

4. Dunk for 1 sec at room temperature in 50 ml 1X Wash Buffer 3. To a 50 ml conical tube, add 5 ml 10X Wash Buffer 3 and 45 ml distilled H2O. Mix thoroughly by vortexing before adding the microarrays.  Transfer microarrays one at a time from Wash Buffer 3 to the 50 ml tube containing Wash Buffer 3.  Dunk each microarray for 1 second to remove Wash Buffer 3 and proceed quickly to the next step. Wash Buffer 3 is designed to remove Wash Buffer 2 components that will cause streaks on the surface if not removed.

5. Spin dry and scan.  Transfer the microarray quickly from Wash Buffer 3 to a Microarray High-Speed Microarray Centrifuge and spin at full speed for 10 seconds to dry the microarray.  The microarray surface should be clean, dry and ready for scanning at this stage.

Figure 1. Wash buffers 1, 2 and 3 are 10X concentrates designed for short oligonucleotide (<25-mer) microarray experiments.  Arrayit buffers provide increased specificity and signal strength, while reducing background.  All buffers arrive pre-mixed and ready to use.

Troubleshooting Tips
High background

• Use Arrayit Blocking Buffers prior to hybridization.
• Increase stringency by elevating temperature of Wash Buffer B or 2.
• Fluorescent probe is drying out during hybridization.  Reduce hybridization time to 4 hours maximum and make sure Cassette is hydrated.

Low signals

• Reduce stringency of Wash Buffer B or 2 by reducing temperature.
• Check probe-labeling efficiency.
• Check mRNA quality.

Scientific Publications
Click on the links for recent scientific publications using Arrayit International, Inc. Arrayit brand products for wash buffer experimentation.

Recommended Products

• Hybridization Cassettes
• High-Throughput Wash Station
• Microarray High-Speed Microarray Centrifuge
• Arrayit Blocking Buffers
• Nanoprint™ Microarrayers
• 946 Microarray Printing Technology
• SpotBot® 2 Personal Microarrayers
• PCR Purification Kits
• InnoScan® Microarray Laser Scanners
• SpotLight™ Microarray Scanners
• SpotWare™ Colorimetric Microarray Scanners