Preparation of Fluorescent DNA Probe from mRNA: Yeast
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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. |
实验频道录入：Protocol 责任编辑：Protocol
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