Preparation of Fluorescent DNA Probe from mRNA: Yeast
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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~undefined | 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 |
| . | ||
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|
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. |
