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Preparation of Fluorescent DNA Probe from mRNA: Yeast

Preparation of Fluorescent DNA Probe from mRNA: Yeast

Last updated: 3/29/99

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.