Mullins Molecular Retrovirology Lab

  • Department of Microbiology
  • School of Medicine
  • University of Washington
University of Washington/Fred Hutch Center for AIDS Research

CY-DYE PROBE Synthesis

Reagents:
Anchored dT25 Amersham, 8 µM, RPK0145, quote 7G-3709, $110
or Anchored dT25 or dT23VN, 8 µM, GibcoBRL
Random 9-mers 5' NNN NNN NNN 3', 1 µg/µl, GibcoBRL
GFP poly A+ RNA prepared from plasmid pSP64-GFP
SuperScript II GibcoBRL, 200 U/µl, 10000 units, 18064-014, $177
5X First Strand Buffer GibcoBRL, included with enzyme
DTT GibcoBRL, included with enzyme
Nucleotides* Pharmacia, 100mM each, 27-2035-02, $00
or Nucleotides* Promega, 10mmol each, U1330, $75
Cy3-dCTP Amersham, 25nmol, PA53023, quote 1A-3709, $151
Cy5-dCTP Amersham, 25nmol, PA55023, quote 1A-3709, $151
RNase Inhibitor Boehringer Mannheim, 40U/ml, 799 017, $00
or Rnasin Promega, 2500 units, N2511, $82
NaOH Sigma, 2.5M, $00
MOPS Sigma, 2M, $00
Glass fiber filter plate Millipore, MAFB NOB 10, 10/pack, $134
Catch Plate VWR, 622409-108, 50/pack, $00
Binding buffer 5.3M Gua-HCl in 150mM Kac pH 4.8 with glacial acetic acid (3-4ml/500ml)
Tris-HCl Sigma, 10mM pH 8.0, T-3038, $00
Ethanol Stores, 80%, $00
ProbeQuant G50 Amersham, 27-5335-01, 50/pack, $145
Coverslips VWR, 24x60mm, 48393-106, $15.92
Deionized Formamide Sigma, 100mL, F-9037, $23.60
20X SSC Ambion, 1L, 9763, $30
50X Denhardt�s Fisher, 500g, BP515-5, $42.40
10% SDS Ambion, 500ml, 9822, $40
Human CotI DNA GibcoBRL, 500 units, 15279-011, $75
Poly A72 5� A72 3�, GibcoBRL
Wash buffers 1XSSC/0.2%SDS, 0.1XSSC/0.2%SDS, 0.1XSSC

* Nucleotide mix: always use 10mM dGTP/dATP/dTTP and 1mM dCTP; unlabeled dCTP used in 1:1 ratio with Cy-labeled dCTP.

CY-DYE PROBE SYNTHESIS (17 February 2001)

Protocol:

  1. Mix together:

2 µg mRNA

2 µl oligo dT23VN (8 µM)

2 µl random 9-mers (1 µg/µl)

1 µl GFP mRNA (1 ng/µl)

10.5 µl total volume

  1. Heat to 70°C for 10 minutes.

  2. Chill on ice for 30 seconds.

  3. Centrifuge briefly.

  4. Add the following:

4 µl 5X First Strand Buffer

2 µl DTT (0.1 M)

1 µl Nucleotide Mix (10 mM G/A/T, 1 mM C)

1 µl Cy3- or Cy5-dCTP (1 mM)

0.5 µl RNase Inhibitor

Note: when doing multiple reactions, make up premix containing all but Cy-dyes and aliquot 7.5 µl premix to each tube before adding Cy-dyes.

  1. Mix contents of tube gently and incubate at RT for 10 minutes.

  2. Add

1 µl SuperScriptII

20 µl total reaction volume

  1. Mix contents of tube gently and incubate at 42°C for 2-3 hours.

  2. Denature cDNA/mRNA hybrid as follows:

Add 2 µl 2.5 M NaOH

Incubate at 37°C for 10 minutes

Add 10 µl 2 M MOPS

  1. Add 200 µl binding buffer to probe and mix.

  2. Dispense into glass fiber filter plate.

  3. Place filter plate on top of vacu-system and apply vacuum.

  4. Wash 6x with 200 ml 80% Ethanol.

  5. Place filter plate on top of catch plate along with centrifuge alignment frame.

  6. Do one dry spin to remove residual ethanol (3500 rpm, 5 min).

  7. Add 50 µl 10 mM Tris-HCl, pH 8. Incubate 1 minute at RT.

  8. Place filter plate on top of a clean catch plate along with a centrifuge alignment frame and spin (3000 rpm, 5 min).

  9. Repeat with another 50 µl Tris-HCl.

  10. Scan probe at OD 260 nm, 550 nm and 650 nm and calculate probe yield, incorporated Cy-dye and specific activity.

  11. Purify probe over G50 column (prespin column (1000x g 1 min), transfer to new tube, apply probe, spin (1000x g 2 min), collect flow-through).

  12. Dry purified probe down in speedvac (1 hour at 50°C).

  13. Rinse slides and coverslips by two quick dips in ddH2O, air dry.

  14. Resuspend dried-down probe in 25 µl volume of hybridization solution (50% formamide, 5x SSC, 5x Denhardt�s, 0.1% SDS, 100 µg/ml poly A72, 100 µg/ml human CotI DNA; pass through 0.2 µm filter).

  15. Boil probe for 3 minutes, ice 30 seconds, spin briefly.

  16. Combine with appropriate reaction; total volume is now 50 µl.

  17. Apply probe to slide and cover with coverslip.

  18. Incubate overnight (14-16 hrs) at 42°C in humid chamber.

  19. Preheat wash buffers to 55°C.

  20. Remove coverslip by immersing slide in 1x SSC/0.2% SDS.

  21. Wash in 1x SSC/0.2% SDS for 10 minutes at RT.

  22. Wash twice in 0.1x SSC/0.2% SDS at RT for 10 minutes.

  23. Wash twice in 0.1x SSC at RT for 1 minute.

  24. Rinse by two quick dips in ddH2O

  25. Dry with compressed air.

  26. Scan (PMT 600, green (532nm), filter 1; PMT650, red (633nm), filter 2; width 10mm, length 60mm).

PS. Name Fluorescence Solution

Cy3 = green = pink

Cy5 = red = blue