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

ArrayIt® advanced 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 Plus was formulated to provide high coupling efficiency and unparalleled spot morphology with oligonucleotides, cDNAs, and other DNA molecules on SuperAmine 2, SuperAldehyde 2, SuperEpoxy 2, SuperMirror 2, and related glass substrate slide surfaces. 50 ml of 2X solution or 50 ml of 4X solution.

Table of Contents
Introduction
Quality Control
Product Description
Technical Assistance
Short Protocol
Complete Protocol
Literature Cited
Equipment 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 Plus.

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 formulation conforms to the highest industry standards.

Product Description
ArrayIt® Micro-Spotting Solution Plus is an advanced buffer system containing a patent-pending mixture of ionic and polymeric materials. Use of our Micro-Spotting Solution Plus will increase the quality of microarrays prepared by all contact and non-contact DNA printing technologies by providing uniform spreading of the sample on microarray surface. Users will appreciate the following features:

• Supports multiple printing technologies
• Reduced background fluorescence than Micro Spotting Solution (MSS)
• Provides uniform and highly regular printed features (spots)
• Facilitates uniform DNA attachment within each spot
• Promotes increased target attachment to the microarray surface
• Slows evaporation of samples within the source microplates
• Enables uniform sample drying
• Reduces or eliminates sample drying in printing pins and ink-jet nozzles
• Washes away easily after printing
• Improves printing consistency
• Expedites data analysis
• Minimizes array-to-array variation
• Sufficient to spot 50,000,000 features
• Stabilizes DNA samples for prolonged storage
• Arrives pre-mixed and sterile, with no preparation required

Figure 1. Samples printed with MSS Plus. A total of 8,000 human cDNAs were purified with TeleChem's PCR Purification Kit, dried to completion and resuspended in 4.0 µl dH₂0. A volume of 4.0 µl of 2X MSS Plus was added to each sample and the samples were mixed thoroughly by pipetting. The samples were printed on a microarray substrate, the printed substrate was processed and hybridized with a our SeeIt Universal Probe at 10 µM concentration in 1X HybIt® Hybridization Solution for 4 hours at room temperature (22°C). The microarray was washed with Wash Buffers 1, 2 and 3 and scanned for fluorescence emission. The quality of the microarrays features and fluorescent signals are observed easily.

Technical Assistance Please contact us if you have any comments or 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 from 8 AM-6 PM Pacific Standard Time (PST). Please remember that we want to hear about your successes! Short Protocol (Steps 1-6) 1. Resuspend cDNAs at 0.25-0.75 µg/µl and oligonucleotides at 30-60 µM. 2. Transfer 4.0 µl of each DNA sample into a 96- or 384-well microplate. 3. Add 4.0 µl per well of 2X ArrayIt® Micro Spotting Solution Plus. 4. Mix the samples thoroughly by pipetting up and down 10 times. 5. Print the DNA samples onto SuperAldehyde, SuperAmine or an equivalent substrate. 6. Process the printed substrates for hybridization. Complete Protocol (Steps 1-6) 1. Resuspend the DNA samples at a cDNA concentration of 0.25-0.75 µg/µl or an oligonucleotide concentration of 30-60 µM. The PCR products (cDNAs) should be purified with the TeleChem PCR Purification Kit (or equivalent) to remove contaminants that may interfere with attachment to the substrate. Oligonucleotides should be free of CPG, ammonium hydroxide and other chemical contaminants derived from phosphoramidite synthesis. All DNA samples (cDNAs and oligonucleotides) should be resuspended at the appropriate concentration in dH20. Users may want to test a range of different concentrations to determine the optical target concentration for a particular assay, though the values given above work very well for many different applications. 2. Transfer 4 µl of each DNA sample into a 96- or 384-well microplate. Sample transfer can be performed manually, with a multi-channel pipetting device, or with a liquid-handling system. Most samples of cDNAs and oligonucleotides pre-exist in a 96-well or 384-well format owing to the fact that many PCR purification and oligonucleotide synthesis schemes use a microplate format. Purification of cDNAs with the ArrayIt® PCR Purification Kits will result in a 96-well or 384-well format for the cDNA samples. Many commercial oligonucleotide synthesis services provide a microplate format as well. Some commercial providers also offer a "buffer of choice" for resuspension, and users can request Micro-Spotting Solution Plus or an equivalent buffer. 3. Add 4.0 µl per well of 2X ArrayIt® Micro-Spotting Solution Plus to each 4.0 µl DNA sample. Pipoetting can be performed manually, with a multi-channel pipetting device or with a liquid-handling system. Make sure that the transfer volume of Micro-Spotting Solution Plus is 4.0 µl ± 10% for all samples, as small differences in the concentration of buffer components can produce variability in spot diameter on the printed microarrays. 4. Mix the DNA samples thoroughly by pipetting up and down 10 times. Micro-Spotting Solution Plus contains a concentrated mixture of ionic and polymeric components, and thorough mixing is required to generate a homogenous sample. Failure to mix the samples thoroughly at this step will result in poor sample loading, inefficient printing and poor microarray quality! Make certain to mix your samples! Sample evaporation can be minimized sealing the microplates with an adhesive seal. Properly sealed plates can be stored for several weeks at 4°C without a significant loss of volume or DNA integrity. Samples can be stored indefinitely at –20°C or –80°C, though samples should be re-mixed after thawing and prior to arraying. 5. Print the DNA samples onto SuperAldehyde or SuperAmine Substrates or onto an equivalent microarray slide by placing the 96-well or 384-well plates on a suitable microarraying device and printing the samples onto the Substrates. The highest quality microarrays are obtained using TeleChem's patented Stealth™ or ChipMaker™ Micro-Spotting Technology fitted to an advanced motion control system such as those provided by Virtek Vision (Ontario, Canada), Cartesian Technologies (Irvine, CA), Packard Biochip Technologies (Billerica, MA), GeneMachines (Belmont, CA), and many other vendors. The optimal printing environment is 20°C and 50-55% relative humidity. For most users, the SuperAmine surface provides the strongest signals and the lowest background across many different applications and hybridization conditions. Superior results can be obtained using SuperAldehyde Substrates, though background tends to increase in cases where technique is less than fastidious. For DNA templates <50 bp or oligonucleotides in the 5-50 nt range, covalent attachment to SuperAldehyde Substrates is strongly recommended as this coupling chemistry favors end-attachment and therefore maximizes the availability of short targets for hybridization. For optimal coupling efficiency on SuperAldehyde Substrates, the use of a 5' amino-linker is recommended strongly. The selectivity of amino-modified versus natural DNA is ~10:1 for cDNAs and ~1,000:1 for single-stranded 15-mers. SuperAmine Substrates provide strong signals and low background for all target lengths >50 bp or 50 nt. Complete protocols are available for the SuperAldehyde and SuperAmine Substrates. 6. After printing, the Substrates should be processed appropriately to allow efficient attachment of the target DNA to the surface. In the case of SuperAldehyde Substrates , an overnight incubation at room temperature (20-30°C) and low humidity (<40%) will facilitate dehydration and Schiff's base formation. The drying step can be allowed to proceed on the platen of the microarrayer or in slide boxes with the lid ajar slightly. Stable attachment to SuperAmine Substrates can be achieved one hour after printing, by drying the Substrates for 80 min at 80°C in a drying oven. Attachment to SuperAmine can be strengthened by subjecting the DNA side of the Substrates to cross-linking with ultraviolet light (e.g. Stratagene Stratalinker). Once the DNA is attached in a stable manner to the substrate, unbound target DNA should be removed and double-stranded DNAs should be denatured. Additional blocking steps can also be used to inactivate unreacted amine and aldehyde groups. Complete processing protocols are available electronically on the Super Microarray Substrates web page. Literature Cited 1. 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. 2. 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. 3. 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. 4. A comprehensive list of nearly 1,000 microarray publications is available electronically at http://arrayit.com/e-library. Requirements Stealth™ or ChipMaker™ Micro Spotting Device PCR Purification Kit High-Throughput Wash Station, HTW SuperAldehyde or SuperAmine Substrates Troubleshooting Tips Poor printing quality: Incomplete mixing of DNA samples and Micro-Spotting Solution Plus Poor Printing Environment: not more than 55% humidity and 22°C recommended Poor DNA attachment on SuperAldehyde substrates: Forgot use amino-modified DNA for SuperAldehyde Substrates Forgot to allow drying overnight at <40% humidity for SuperAldehyde Substrates Forgot to stabilize DNA on SuperAmine by baking at 80°C for 80 min Sample contaminants prevent efficient attachment Elevated background fluorescence: Poor slide processing Poor purification of probe prior to hybridization. ArrayIt® Fluorescent Probe Purification kit is recommended.