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

 

 2. Heat to 70°C for 10 minutes.

 3. Chill on ice for 30 seconds.

 4. Centrifuge briefly.

 5. 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.

 

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

 7. Add

          1 µl SuperScriptII

         20 µl total reaction volume

 

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

 9. 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

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

 11. Dispense into glass fiber filter plate.

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

 13. Wash 6x with 200 ml 80% Ethanol.

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

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

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

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

 18. Repeat with another 50 µl Tris-HCl.

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

 20. 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).

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

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

 23. 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).

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

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

 26. Apply probe to slide and cover with coverslip.

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

 28. Preheat wash buffers to 55°C.

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

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

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

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

 33. Rinse by two quick dips in ddH2O

 34. Dry with compressed air.

 35. 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

Department of Microbiology
School of Medicine
University of Washington
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