Micro Spotting Solution, 50 ml of 4X

Arrayit Micro Spotting Solution, 50 ml of 4X is the original microarray printing 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. Arrayit MSS 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, Mirror, and related glass substrate slide surfaces, 50 ml of 4X solution.

Buffers and Solutions - Micro Spotting Solutions to Enhance the Efficiency of DNA Microarray Manufacturing by Contact Printing for Research, Life Sciences, Agricultural and Microarray Testing Applications

Arrayit offers the original microarray spotting buffers 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. Arrayit Micro Spotting Solutions were developed to provide high coupling efficiency and good spot morphology with oligonucleotides, cDNAs and other DNA molecules on SuperAmine, SuperAldehyde, SuperEpoxy, Mirror, and related glass substrate slide (1 x 25 x 76 mm) surfaces. Available in 2X and 4X formulations, 50 ml per bottle.

Table of Contents

• Introduction
• Quality Control
• Product Description
• Short Protocol
• Complete Protocol
• Literature Cited
• Equipment Requirements
• Troubleshooting Tips
• Technical Support
• Scientific Publications
• 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 Solutions.

Quality Control
Arrayit assures the performance of this product line. The finest scientific research was used to develop this product line. Rigorous quality control monitoring on a lot-by-lot basis guarantees that the ingredients conform to the highest industry standards.

Product Description
Arrayit Micro Spotting Solutions are advanced buffers containing a proprietary mixture of ionic and polymeric materials. Arrayit Micro Spotting Solutions 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 features

Figure 1. Arrayit Micro Spotting Solution (Cat. MSS) Improves Microarray Performance. Shown are two scanned images of microarrays printed with DNA samples diluted in 5X SSC (top) or Arrayit Micro Spotting Solution (bottom). Arrayit ChipMaker™ printing technology was used to print the samples at 150 µm center-to-center spacing onto silylated slides. Fluorescent detection of the Cy3-labeled hybridization was performed using the ScanArray 3000 from General Scanning, Inc. Improvements in microarray quality are easily observed by using Arrayit Micro Spotting Solution.

Short Protocol (Steps 1-7)
1. Attach a 5’ amino-linker group to the 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 2X 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. 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 microarray surface, the covalent amino-modification is stable to a wide range of temperatures and solvents. The 5’end attachment of the DNA to the microarray surface 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 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 2X Arrayit Micro Spotting Solution (Cat. MSS) or 2.0 µl per well of 4X Arrayit Micro Spotting Solution (Cat. MSS4X) with a multi-channel pipetting device.

5. Mix the amino-modified DNA and the Arrayit Micro Spotting Solution thoroughly by pipetting up and down 10 times. Arrayit 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 reduced microarray quality.

6. Print the amino-modified DNA samples onto silylated microscope slides or Arrayit SuperEpoxy or SuperAldehyde by placing the 96-well or 384-well plates on a suitable microarray printing 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 Arrayit Stealth, 946 or Pro printing technology 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.

Equipment Requirements
SpotBot 4 or NanoPrint 2 Microarrayers
Arrayit Microarray Printing Technology
High-Throughput Wash Stations
Arrayit Microarray Substrate Slides

Troubleshooting Tips

• Reduced printing quality:
• Incomplete mixing of DNA samples and Arrayit Micro-Spotting Solution
• Reduced DNA attachment:
• Make sure to use amino-modified DNA and silylated slides
• Elevated background fluorescence:
• Reduced slide processing efficiency
• Contaminants in labeling reaction. Make sure to use Arrayit Fluorescent Probe Purification Kits