title: Duke University Murine Operon v.2.0 Spotted Array
status: Public on Dec 06 2005
contact.city: Durham
contact.first: Zachary
contact.middle: Conrad
contact.last: Hartman
contact.email: zch@duke.edu
contact.state: NC
contact.address: Research Drive MSRB rm 414
contact.department: Surgery
contact.country: USA
contact.institute: Duke University
contact.phone: 919-684-9197
organism: Mus musculus
description: Protocol for Preparing Poly-Lysine Slides for Microarrays page 1 Basics: -The following protocol is for coating 180 slides with poly-l-lysine. -Be sure slide racks are bent slightly inwards in the middle to hold slides more securely. -DO NOT use powdered gloves at any time during this protocol. -To avoid dust, try to keep slides covered or submerged in solution at all times. -It is recommended that at least two batches of poly-lysine slides are prepared per printing. 1. Prepare wash solution for 180 slides: 200 g NaOH pellets 1200 ml 95% EtOH 800 ml H2O 2. Mix wash solution until NaOH pellets are completely dissolved. 3. Rinse slide dishes completely with H2O. Add one rack of Gold Seal Microslides(30 slides/rack) per slide dish. Pour wash solution over slides and cover. 4. Shake slides gently for 2 hours in wash solution. Although the slides come “pre-washed” the are still significant amounts of oils and debris. This basic wash is necessary to completely clean the slides before applying the poly-lysine. 5. Rinse slides with several liters of Milli-Q H2O. I like to fill a large container with H2O so that I can vigorously dump the rinse over the slides. Let the slides sit in the water while you prepare the poly-lysine solution. 6. Prepare poly-lysine solution (**use only plastic-ware when preparing poly-lysine): 180 slides: 1500 ml H2O 205 ml 1XPBS (filtered) 352 ml poly-l-lysine 7. Mix ingredients on stir plate in plastic beaker in order listed above. 8. Before adding poly-lysine, dump H2O from slides. Immediately after adding poly-lysine pour solution over slides. 9. Let slides shake gently in poly-lysine for 30 minutes. 10. Rinse slides again with Milli-Q H2O in same fashion as described above. 11. Immediately spin slides in tabletop at 600 rpm until dry. 12. To ensure slides are completely dry, place racks in 50°C vacuum oven for at least 5 min. Protocol for Preparing Poly-Lysine Slides for Microarrays page 2 13. Store slides in a clean plastic slide box. Slides MUST be stored for at LEAST 14 days before spotting DNA. We do not recommend using slides that are more than 4 months old as degradation of poly-lysine has been observed. **Poly-lysine solution can be reused if you expect to prepare a large volume of slides. To do so, filter lysine solution after step 9 and store at 4°C in a plastic container. When ready to use, distribute solution to slide dishes and add an additional 10-15 ml of new poly-lysine to each dish. This should be repeated a maximum of 6 times. After slides are preparred they are spotted with Operon olgionucleotides, for details see weblink below.
manufacture_protocol: The Operon mouse oligonucleotide library,version 3.0, comprised of 70-mer oligonucleotides representing 24,878 unique mouse genes, was printed on poly-L-lysine-coated glass slides. The library also included sets of positive and negative controls that were used for quality control purposes. Oligonucleotides were spotted using a GeneMachines Omnigrid arrayer by the Duke University Microarray facility. Details can be found at http://mgm.duke.edu/genome/dna_micro/core/spotted.htm.
  