Micro Spotting Solution

Products - Buffers & Solutions - Microarray Print Buffers - Micro Spotting Solution

ArrayIt® offers the original microarray print buffer containing a mixture of viscosity enhancers, stabilizers, and buffering components to increase the quality and stability of DNA microarrays by improving the surface properties of the DNA samples deposited during printing. Micro Spotting Solution was developed to provide high coupling efficiency and good spot morphology with oligonucleotides, cDNAs, and other DNA molecules on SuperAmine, SuperAldehyde, SuperEpoxy, SuperMirror, and related glass substrate slide surfaces. For applications requiring ultra-low background, use Micro Spotting Solution Plus. 50 ml of 2X solution.

Table of Contents
Introduction
Quality Control
Product Description
Technical Assistance
Short Protocol
Complete Protocol
Literature Cited
Requirements
Troubleshooting Tips
Ordering Information
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^® Micro Spotting Solution.

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 Description
The ArrayIt^® Micro Spotting Solution is an advanced buffer system containing a patent-pending mixture of ionic and polymeric materials. Use of our Micro Spotting Solution will increase the quality of microarray biochips prepared by all direct surface contact DNA printing technologies.

Users will appreciate the following features:

Supports multiple printing technologies
Improves micro-spotting consistency (<0.01% feature loss)
Increases sample surface tension which reduces feature size
Improves deposition precision allowing easier data analysis
Increases deposition uniformity which improves quantitation
Greater feature uniformity minimizing grid-to-grid variation
Buffer components wash away during processing
Stabilizes DNA samples for prolonged storage
Arrives pre-mixed and sterile, no preparation required
Sufficient to spot 50 million feature

Figure 1. Effect of Micro Spotting Solution on microarray quality. Shown are two scanned images of microarrays printed with DNA samples diluted in 5X SSC (left) or TeleChem’s ArrayIt® Micro Spotting Solution (right). The ChipMaker™ Micro Spotting device was used to print the samples at 150 µm center-to-center spacing onto silylated slides. Fluorscent detection of the Cy3-labeled hybridization probe was performed using the ScanArray 3000 from General Scanning, Inc. Improvements in microarray quality are easily observed by using ArrayIt® Micro Spotting Solution.

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!

Short Protocol (Steps 1-7)
1. Attach covalently a 5’ amino-linker group to DNA samples.
2. Resuspend amino-modified DNA samples in H₂0 at the desired concentration.
3. Transfer 4.0 µl of each DNA sample into 96-well or 384-well microplates.
4. Add 4.0 µl per well of ArrayIt® Micro Spotting Solution.
5. Mix the samples by pipetting up and down 10 times.
6. Print the amino-modified DNA samples onto silylated microscope slides.
7. Process the slides for hybridization.

Complete Protocol (Steps 1-7)
1. Covalently attach a 5’ amino-linker to oligonucleotides or PCR products either by modification of the oligonucleotides directly during oligonucleotide synthesis or by enzymatic incorporation of amino-modified PCR primers into cDNAs during PCR amplification. The 5’ amino-modification used most successfully with our surface chemistry is the NH2(CH2)6 linker from Glen Research. The 5’ amino-linker allows selective binding of the amino-containing DNA to silylated slides through a Schiff’s base reaction with aldehyde groups on the chip surface. The selectivity of amino-modified versus natural, unmodified DNA is ~10:1 for cDNAs and ~10,000:1 for single-stranded 15-mers. DNA molecules of intermediate lengths exhibit intermediate discrimination ratios. Once bound to the chip surface, the covalent amino-modification is stable to a wide range of temperatures and solvents. The 5’end attachment of the DNA to the chip via the amino group permits steric accessibility of the bound molecules during the hybridization reaction. The 5’ amino-modification does not appreciably change the solubility of the DNA (i.e. oligos and cDNAs with amino-linkages have solubilities comparable to natural, unmodified DNA.

2. Re-suspend the DNA samples containing a 5’ amino modification in dH20 at the desired concentration. For PCR products to be used for gene expression monitoring, a amino-modified DNA concentration of 0.2-1.0 micrograms per microliter is ideal. For 15-mer oligonucleotides to be used in mutation detection, an amino-modified DNA concentration of 10-100 pmole/µl is ideal.

3. Transfer the amino-modified DNA samples into 96 well or 384 well plates. This is best performed using a multi-channel pipetting device. Purification of cDNAs with the ArrayIt® PCR Purification Kits will result in a 96 well or 384 well format for the cDNA samples. If oligonucleotides are synthesized in a 96 well format, the oligonucleotides may be obtained commercially in a 96 well or 384 well format.

4. Once the amino-modified DNA samples are transferred to a 96-well or 384-well format, add 4.0 µl per well of ArrayIt® Micro Spotting Solution with a multi-channel pipetting device.

5. Mix the amino-modified DNA and the Micro Spotting Solution thoroughly by pipetting up and down 10 times. The Micro Spotting Solution contains a concentrated mixture of ionic and polymeric components and thorough mixing is required prior to DNA printing. Failure to mix the samples thoroughly at this step will result in poor microarray quality!

6. Print the amino-modified DNA samples onto silylated microscope slides by placing the 96 well or 384 well plates on a suitable microarraying device. Sample evaporation can be minimized by placing wetted filter discs (e.g. Whatman) on the underside of the microplate lid and sealing the microplates with flexible laboratory film (e.g. Parafilm) before and after printing. Properly sealed plates containing wetted filter discs can be stored for several weeks at 4°C without detectable loss of volume or DNA stability. For best results, use the ArrayIt® Stealth or ChipMaker™ Micro Spotting device for high-density DNA printing.

7. Following printing, the slides should be left at room temperature for 24 hrs to permit thorough drying of the DNA onto the surface of the silylated slides. This can be accomplished by placing the slides in a slide box with the lid slightly ajar. Direct open air drying of the slides is not recommended as dust and debris will accumulate on the microarray surface. Following the 24 hr drying period, the region on the slide containing the microarray should be marked on the underside to facilitate the downstream hybridization and detection steps. This can be accomplished by lightly scoring the underside of the slide with a diamond pencil. Do not score the DNA side of the slide. Slides should be processed to remove unbound DNA and the components of the Micro Spotting Solution. Many protocols have been used successfully for the slide processing step. One protocol is given below. Load six printed, dried and scored slides into the ArrayIt® Wash Station. Transfer the Wash Station and six slides to a 600 ml beaker containing a stir bar and wash with vigorous agitation with the following solutions. Twice in 0.2% SDS at 25°C for 5 min each, twice in dH20 at 25°C for 5 min each, once in dH20 at 95°C for 2 min, cool to 25°C for 5 min, once in sodium borohydride solution (1.3 g Na2BH4 dissolved in 375 ml phosphate buffered saline, then add 125 ml pure ethanol) at 25°C for 5 min, three times in 0.2% SDS for 1 min each, twice in dH20 at 25°C for 1 min each. Air dry the slides to completion. Slides are ready for hybridization.

Literature Cited
J. Lamture, K.L. Beattie, B.E. Burke, M.D. Eggers, D.J. Ehrlich, R. Fowler, M.A. Holis, B.B. Kosicki, R.K. Reich, S.R. Smith, R.S. Varma and M.E. Hogan (1994). Direct detection of nucleic acid hybridization on the surface of a charge coupled device. Nucl. Acids Res. 22, 2121-2125.

Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P.O., and R.W. Davis (1996). Parallel Human Genome Analysis: Microarray-Based Expression Monitoring of 1,000 Genes. PNAS 93, 10614-10619.

Heller, R.A., Schena, M., Chai, A., Shalon, D., Bedilion, T., Gilmore, J., Woolley, D.E., and R.W. Davis (1997). Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. PNAS 94, 2150-2155.

Requirements
’5-Amino-Modifier C6 (Cat.# 10-1906), Glen Research, Sterling, VA
ChipMaker or Stealth Micro Spotting Device
Wash Station (Cat.# AWS)
SuperAldehyde Substrates

Troubleshooting Tips

Poor printing quality:

Incomplete mixing of DNA samples and Micro-Spotting Solution

Poor DNA attachment:

Forgot to use amino-modified DNA and/or silylated slides

Elevated background fluorescence:

Poor slide processing
Contaminants in labeling reaction use Fluorescent Probe Purification Kit

Ordering Information

Product

Quantity

Catalog ID

Price (US dollars)*

Micro Spotting Solution 2X

ArrayIt® original microarray print buffer containing a mixture of viscosity enhancers, stabilizers, and buffering components to increase the quality and stability of DNA microarrays by improving the surface properties of the DNA samples deposited during printing. Micro Spotting Solution was developed to provide high coupling efficiency and good spot morphology with oligonucleotides, cDNAs, and other DNA molecules on SuperAmine, SuperAldehyde, SuperEpoxy, SuperMirror, and related glass substrate slide surfaces. For applications requiring ultra-low background, use Micro Spotting Solution Plus. 50 ml of 2X solution.

MSS

$126.50

Micro Spotting Solution 4X

ArrayIt® original microarray print buffer containing a mixture of viscosity enhancers, stabilizers, and buffering components to increase the quality and stability of DNA microarrays by improving the surface properties of the DNA samples deposited during printing. Micro Spotting Solution was developed to provide high coupling efficiency and good spot morphology with oligonucleotides, cDNAs, and other DNA molecules on SuperAmine, SuperAldehyde, SuperEpoxy, SuperMirror, and related glass substrate slide surfaces. For applications requiring ultra-low background, use Micro Spotting Solution Plus. 50 ml of 4X solution.

MSS4X

$181.50

Discounts:
1-4 bottles = List price
5-9 bottles = 5% discount
10+ bottles = 25% discount

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

*To order ArrayIt® brand products: call 408-744-1331, fax 408-744-1711 or click on the Purchase buttons to proceed directly to the purchase page.

Warranty
ArrayIt® brand products have been developed scientifically, and are sold for research purposes. Extreme care and exact attention should be practiced in the use of the materials described herein. ArrayIt® brand products are subject to extensive quality control and are guaranteed to perform as described when used properly. Any problems with any 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 is the full responsibility of the user, and TeleChem makes no warranty or guarantee under such circumstances.