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.
Support
glass
Coating
polysine
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.
Expression data from an involution time course in wild type and E2F3 +/- mice.
Data table header descriptions
ID
Oligo_Type
C-Oligo represents all transcripts of an Ensembl gene, P-Oligo represents a subset of transcripts of an Ensembl gene, I-Oligo represents one transcript of an Ensembl gene, M-Oligo represents multiple Ensembl genes
GENE_ID
Operon OMAD Gene Idenification number, www.operon.com/arrays/omad.php?
TRANSCRIPT_COUNT
Number of unique transcripts probe matches
REPRESENTATIVE_TRANSCRIPT_ID
Operon OMAD Representative Transcript ID, www.operon.com/arrays/omad.php?
MarkerSymbol
Common Gene Name
GB_ACC
Genbank or RefSeq Accession Number
GB_ACC
DESCRIPTION
Gene Description
SPOT_ID
Data table
ID
Oligo_Type
GENE_ID
TRANSCRIPT_COUNT
REPRESENTATIVE_TRANSCRIPT_ID
MarkerSymbol
GB_ACC
GB_ACC
DESCRIPTION
SPOT_ID
M200004485
I
ENSMUSG00000000001
1
ENSMUST00000000001
Gnai3
U38502
GUANINE NUCLEOTIDE-BINDING PROTEIN G(K), ALPHA SUBUNIT (G(I) ALPHA-3). [Source:SWISSPROT;Acc:Q9DC51]
SEX COMB ON MIDLEG HOMOLOG 1; SEX COMB ON MIDLEG-LIKE 1 (DROSOPHILA); SEX COMB ON MIDLEG-LIKE 3 (DROSOPHILA); EXPRESSED SEQUENCE AI315320. [Source:RefSeq;Acc:NM_013883]
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