title: Duke University Murine Operon v.3.0 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.