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| Materials for 2 reactions (Cy3 & Cy5) | Qty | Order info | Heat blocks, 42C & 70C | 1 each | Oligo dT (2 ug/uL) | 5.0 uL | Superscript II RT kit | Gibco BRL #18064-014 | 50X dNTPs (25 mM; 10 mM dTTP)* | 1.25 uL | Cy3-dUTP (1 mM) | 3.0 uL | Amersham #53022 | Cy5-dUTP (1 mM) | 3.0 uL | Amersham #55022 | Microcon-30 filter | 3 | Amicon #42410 | TE (pH 7.4) | ~5 mL | polyA DNA or RNA (10 mg/mL in TE) | 1.0 uL | 20X SSC | 3.0 uL | Millipore filter, 0.45 um | 1 | Millipore #UFC3OHVNB | . | *50X dNTPs stock | Final conc. | dATP (100 mM) | 25 uL | 25 mM | dCTP (100 mM) | 25 uL | 25 mM | dGTP (100 mM) | 25 uL | 25 mM | dTTP (100 mM) | 10 uL | 10 mM | ddH2O | 15 uL | --------- | 100 uL | |
| 1. | PREPARE PRIMER-ANNEALED RNA: | Cy3 rxn | Cy5 rxn | RNA (2 ug mRNA or 15 ug total) | 12.9 (RNA #1) | 12.9 (RNA #2) | Oligo dT (2 ug/uL) | 2.5 | 2.5 | --------- | --------- | 15.4 uL | 15.4 uL | Heat 10 min. @ 70C. Quick chill on ice. | 2. | PREPARE RT COCKTAIL FOR BOTH RXNS: | X 1 | X 2.5 | Final conc. | 5X Superscript II buffer | 6.0 | 15.0 | 1X | DTT (0.1 M) | 3.0 | 7.5 | 10 mM | 50X dNTPs | 0.6 | 1.25 | 500 uM; 200 uM dTTP | Cy3- or Cy5 dUTP | 3.0 | --- | 100 uM | Superscript II (200 U/uL) | 2.0 | 5.0 | 13 U/uL | --------- | --------- | 14.6 uL | 11.6 uL aliquots | |
| 3. | Add 3.0 uL Cy3 or Cy5 to respective primer-annealed RNAs. | ||||||||||||||||||||||||||
| Aliquot 11.6 uL of RT cocktail to each rxn for total volume of 30 uL. | |||||||||||||||||||||||||||
| Incubate 2 hr @ 42C. Place on ice. | |||||||||||||||||||||||||||
| 4. | Place 500 uL TE (pH 7.4) each in two microcon-30 filters. | ||||||||||||||||||||||||||
| Add RT rxns to each microcon filter. Centrifuge 7 min. at top speed. | |||||||||||||||||||||||||||
Optional: Repeat TE washes 1-2 times, or until all unincorporated dye is removed. | 5. | Inspect filters. Centrifuge in 30 sec. intervals until volume is 10-20 uL. | 6. | Invert filters into fresh tubes. Centrifuge 1 min. to harvest labeled cDNA. | Optional: For next day hybridization, store cDNA @ 4C. | 7. | Mix together Cy3- and Cy5-labeled cDNAs. | Concentrate sample to ~10-12 uL using microcon filter or vacuum pump. | 8. | Add 1 uL polyA DNA or RNA for non-specific hybridization. | Add 3 uL 20X SSC for total volume of 12-15 uL. | 9. | Pre-wet millipore filter by adding 5 uL ddH2O. Centrifuge 1 min. at top speed. | Remove eluted water with pipet tip. | 10. | Add probe to filter. (Pipet probe onto filter wall, not directly onto membrane.) | Centrifuge 1 min. at top speed. | => | PROBE IS NOW READY FOR HYBRIDIZATION. | REMEMBER TO ADD 0.3 uL SDS (10%) TO PROBE as stated in hybridization protocol. | |