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.
