SCHOTT Nexterion Slide A User manual

Protocol Nexterion™ Slide A
Short Version
1
Technical Instructions for Spotting Microarrays
PRODUCT
NexterionSlide A is an activated glass slide in the standard size 75.5 mm x 25 mm x 1.0
mm with a multi-amino-silane* surface coating for efficient binding of cDNA molecules, PCR
products and longer oligonucleotides (size ≥50 mers).
*patent pending
DURABILITY AND STORAGE
Use by shelf-life date if stored in sealed condition. Open and use the slides in a clean
environment to avoid particle build-up on the printing surface. Avoid direct contact with the
printing surface to minimize contamination and abrasion of the coated surface. After opening
the package, store the remaining slides in a dust-free, light protected, and dry area at room
temperature and use within two weeks.
ARRAY PRINTING
1. Mix equal amounts of oligonucleotide probe or PCR product and 50% DMSO to obtain
a minimum final probe concentration of 20 µM for oligos, or 0.3 mg/ml for PCR
products. For smaller spot sizes, 3X SSC can be used as a printing buffer.
2. Transfer an appropriate volume of probes to a microtiter plate.
3. Set up the arrayer according to the manufacturer’s recommendation and print slides at
40-50% relative humidity.
DNA IMMOBILIZATION
1. Rehydrate slides over a boiling water bath for 2-3 seconds, and then snap-dry for 10
seconds at 85°C on a hot plate.
2. UV cross link at 600 mJ for PCR products or 800 mJ for oligonucleotides.
3. Proceed immediately to prehybridization (washing and blocking) or store the arrays in
a desiccator for no more than two weeks.
<OPTIONAL> ARRAY DENATURATION (only for PCR products)
1. 1 x 30 sec in 0.1% SDS at room temperature.
2. 1 x 3 min in deionised H2O at 95°C.
3. 1 x 2 min 70% ethanol.
4. Spin-dry slides immediately at 200 x g for 5 min.
WASHING AND BLOCKING
1. 1 x 60 min Prehybridization Buffer (5X SSC, 0.1% SDS, 1.0% BSA, 50% formamide,
0.01% salmon sperm DNA) at 42°C.
2. 5 x deionised H2O at room temperature.
3. Spin-dry slides immediately.

Protocol Nexterion™ Slide A
Short Version
2
HYBRIDIZATION
1. Re-suspend or dilute the labeled target in Prehybridization Buffer (5X SSC, 0.1% SDS,
1.0% BSA, 50% formamide, 0.01% salmon sperm DNA).
2. Denature the suspended target by heating at 95°C for 3 min and apply the appropriate
volume onto the array surface of a blocked slide.
POST-HYBRIDIZATION AND WASHING
1. Wash 1 x 5 min in 2X SSC and 0.1% SDS at room temperature to remove the cover
slip.
2. Wash 1 x 5 min in 2X SSC at room temperature.
3. Wash 1 x 1 min in 0.2X SSC at room temperature.
4. Wash 1 x 5 sec in 0.05X SSC at room temperature.
5. Dry the array in an oil free air or nitrogen stream or by centrifugation.
IMPORTANT INFORMATION ABOUT PATENTS
Using arrays based on SCHOTT Nexterion products for dual color analysis on a single array in
which at least two different samples are labeled with at least two different labels may require a
license under one of the following patents: U.S. patent nos. 5,770,358 or 5,800,992 or
6,225,625 and U.S. patent no. 5,830,645. Manufacturing and use of probe arrays may require
a license under the following patents: U.S. patent no. 6,040,138 or 5,445,934 or 5,744,305
and under the following patents owned by Oxford Gene Technology Ltd. („OGT“): European
patent no. EP 0,373,203, U.S. patent nos. 5,700,637 and 6,054,270 and Japanese patent nos.
3393528 and 3386391 ("The OGT patents").
Other patents may apply. The purchase of NexterionTM products does not convey any license
under any of the OGT patents or any of the other patents referred to. For all applications
SCHOTT North America Inc. and SCHOTT Nexterion AG make no representation or warranty
that the practice of its technology and products or any improvement will not infringe or violate
any domestic or foreign patent of any third party. To inquire about licensing under the OGT
patents, please contact OGT at [email protected].
Complete protocol, additional information and online-ordering at:
USA/Can: www.us.schott.com/nexterion
Europe/Asia: www.schott.com/nexterion
For technical assistance, please contact
Europe / Asia – Pacific: USA / Canada:
Schott Nexterion AG Schott Nexterion
Winzerlaer Str. 2a a Division of Schott North America Inc.
07745 Jena 400 York Avenue
Germany Duryea, PA 18642
Phone: [phone] USA
Fax: [phone] Phone: [phone], x657
E-Mail: [email protected] Fax: [phone]
E-Mail: [email protected]
© February 2004