John M. S. Bartlett.pdf - Bio-Nica.info

246 Ringquist et al.
In this chapter, protocols are presented for the synthesis of RAP-PCR probes for
application to microarrays. Arrays spotted on nylon membranes can also be used.
The manufacture of microarrays, themselves, is discussed only briefly, and the
reader is reminded that there are a number of different approaches. For example,
diverse chemistries for adhering cDNA sequences to glass slides, PCR-amplified vs.
synthesized spotted sequences, different fluorescent dye-labeled nucleotides, different
robotic spotting strategies, and other variables have been explored. The reader is
encouraged to seek current technical information in this rapidly moving field.
2. Materials
2.1. Special Reagents and Supplies
1. RNeasy Mini Kit (Qiagen #74106, Valencia, CA).
2. QIAshredder columns (Qiagen #79656, Valencia, CA).
3. QIAquick PCR Purification Kit (Qiagen, #74106, Valencia, CA).
4. Microcon YM-30 (Millipore #42410, Bedford MA).
5. Human cDNA clones (I.M.A.G.E; Research Genetics, Huntsville, AL).
6. Vector-specific primers (Genosys Biotechnologies, The Woodlands, TX): Forward:
5′-ctgcaaggcgattaagttgggtaac-3′, Reverse:5′-gtgagcggataacaatttcacacaggaaacagc-3′
7. First strand cDNA primer: oligo(dT) 20 .
8. Arbitrary primers: primer A: (5′-ACGAAGAAGAAGAG), primer B (5′-GTGACAGACA;
Genosys Biotechnologies, The Woodlands, TX).
9. [α- 32 P] dCTP 10 Ci/mL (ICN, Irvine, CA).
10. Random hexamers (NNNNNN; Genosys Biotechnologies, The Woodlands, TX).
11. Cy3-dCTP and Cy5-dCTP (Amersham, Piscataway, NJ).
2.2. Cell Culture
1. Fibroblast (ATCC #CRL 2091, Manassas, VA).
2. Cell-culture dishes (150 cm; Nunc, Rochester, NY).
3. Cell culture media (DMEM, Irvine Scientific #9031 Irvine, CA), plus 10% fetal bovine
serum (Omega scientific #FB-01, Tarzana, CA).
4. Penicillin (1000 U/mL).
5. Streptomycin (1 mg/mL).
2.3. Enzymes
1. M-MLV reverse transcriptase (200 U/µL; Promega, Madison, WI).
2. AmpliTaq DNA polymerase Stoffel fragment (10 U/µL; Perkin–Elmer Cetus, Norwalk,
CT).
3. RNase-free DNase (10 U/µL) (Roche Molecular Biochemicals, Indianapolis, IN).
4. RNase inhibitor (40 U/µL) (Roche Molecular Biochemicals, Indianapolis, IN).
2.4. Common Reagents, Supplies, Buffers, and Equipment
1. DMSO.
2. Distilled, deionized H 2 O.
3. 1× TE (pH 8.0).
4. Formamide dye solution.
5. 4% polyacrylamide, 8 M urea sequencing-style gels, prepared with 1× TBE buffer.
6. 1% agarose minigels (1× TBE).
7. 5× DNase I digestion buffer: 100 mM Tris-HCl, 50 mM MgCl 2 , pH 8.0.