30 May 2002

Reagents:

Oligo-dT/T7 (Eberwine) 5�-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T15-3�
 or T7-oligodT (Luo) 5�-TCT AGT CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GCGT21-3�
SuperScript II GibcoBRL, 200 U/µl, 18064-022
5X First Strand Buffer GibcoBRL, included with enzyme
DTT GibcoBRL, 0.1 M, included with enzyme
Nucleotides Pharmacia, 100 mM each of dGTP, dATP, dTTP, dCTP, 27-2035-02* 
RNase Inhibitor Boehringer Mannheim, 40 U/µl, 799017
 or RNasin GibcoBRL, 40 U/µl15518-012
Linear Acrylamide Ambion, 0.1 µg/µl, 9520
5x Second Strand Buffer 100 mM Tris pH 6.9, 450 mM KCl, 23 mM MgCl2, 750 µM β-Nicotine Adenine Dinucleotide (Sigma, N6522), 50 mM (NH4)2SO4
E. coli DNA polymerase I NEB, 10 U/µl, 209L or GibcoBRL, 10 U/µl, 18010-017
E. coli RNase H GibcoBRL, 2 U/µl, 18021-071
E. coli DNA ligase NEB, 10 U/µl, 205L or GibcoBRL, 10 U/µl, 18052-019
Phenol:Chloroform:IAA Ambion, 25:24:1, pH 6.6, raised to pH 7.9 with included buffer (400 ml), 9732
Ammonium Acetate Ambion, 5M (0.2 µm), 9070G
Ethanol Stores, 100% (-20°C)
T7 MegaScript Kit Ambion, 1334
 or Ampliscribe T7 Kit Epicentre, AS2607 (25x)/AS3107 (50x)
MicroBioSpin column BioRad, P-6 732-6221, P-30 732-6223
Phase-Lock Gel Light Brinkman Instruments, 1.5 ml, 0032007.961

* Nucleotide mix: use 10 mM dGTP/dATP/dTTP/dCTP.


Protocol:

  1. Mix together:

                       200 ng       poly A+ RNA (or 4-10 µg total RNA)

                           1 µl       oligo-dT/T7 (1 µg/µl)

                         10 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 (on ice):

                       4 µl         5x First Strand Buffer

                       2 µl         DTT (0.1 M)

                       1 µl         dNTP mix (10 mM)

                       1 µl         Linear Acrylamide (0.1 µg/µl)

                       1 µl         RNase Inhibitor (40 U/µl)

    Note: when doing multiple reactions, make up premix containing all and aliquot 9 µl premix to each tube.

  6. Mix contents of tube gently. Heat to 42°C for 1 minute.

  7. Add:

                          1 µl         SuperScript II (200 U/µl)

                        20 µl         total volume

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

  9. Place on ice and keep cool while adding second strand components.

  10. Add the following:

                        30 µl         5x Second Strand Buffer (recipe below)

                          3 µl         dNTP mix (10 mM)

                          4 µl         E. coli DNA polymerase I (10 U/µl)

                          1 µl         E. coli RNase H (2 U/µl)

                          1 µl         E. coli DNA ligase (10 U/µl)

                        91 µl         ddH2O

                       150 µl        total volume

  11. Mix contents of tube gently and incubate at 16°C for 2 hours.

  12. Add 3 µl 2.5M NaOH and incubate at 37°C for 10 minutes.

  13. Add 150 µl buffered Phenol:Chloroform:Isoamylalcohol (25:24:1).

  14. Mix by pipetting, spin 15K for 5 minutes.

  15. Transfer aqueous phase to new tube.

  16. Precipitate with 150 µl 5 M Ammonium Acetate and 1 ml 100% Ethanol.

  17. Keep at -20°C for at least 15 minutes.

  18. Vortex, spin 15K for 20 minutes at RT.

  19. Add 100 µl 100% Ethanol, spin 15K for 5 minutes.

  20. Resuspend dry pellet in 50 µl ddH2O or 10mM Tris pH 7.5.

  21. Pass through BioRad MicroBioSpin column (P-6 > 5bp, P-30 > 20bp). Mix column, remove cap, snap off bottom, spin 2 minutes at 1000x g, change collection tube, add sample, spin 4 minutes at 1000x g.

  22. Dry purified probe down in speedvac to 16 µl or less.

  23. T7 amplification kit: use all 16 µl in 40 µl reaction (double volume). Add sequentially (at RT):

                     16 µl         template DNA

                       4 µl         10x T7 Reaction Buffer

                     12 µl         NTP mix (100 mM)

                       4 µl         DTT (100 mM)

                       4 µl         Ampliscribe T7 enzyme solution

                     40 µl         total volume

  24. Incubate 42°C for 2-3 hours.

  25. Add 110 µl H2O and 150 µl water-saturated Phenol:Chloroform:Isoamylalcohol (25:24:1).

  26. Mix by pipetting, spin 15K for 5 minutes.

  27. Transfer aqueous phase to new tube.

  28. Precipitate with 150 µl 5 M Ammonium Acetate and 1 ml 100% Ethanol.

  29. Keep at -20°C for at least 15 minutes.

  30. Vortex, spin 15K for 20 minutes at RT.

  31. Add 100 µl 100% Ethanol, spin 15K for 5 minutes at 4°C.

  32. Resuspend dry pellet in 30-50 µl ddH2O.

  33. Pass through BioRad MicroBioSpin column as in step 21.

  34. Quantitate approximate aRNA yield by Bioanalyzer and spectrophotometer (1 µl).

Ready for CyDye probe synthesis. Label at least 5 µg of amplified RNA per reaction with 3-8 µg random hexamers. Yield ~20 µg aRNA from 200ng mRNA.


5x Second Strand Buffer Recipe:

Final Concentration                 Stock         10 ml

100 mM Tris pH 7.0              1.0 M         1.000 ml

450 mM KCl                         2.0 M         2.250 ml

23 mM MgCl2                       1.0 M         0.230 ml

50 mM (NH4)2SO4               1.0 M         0.500 ml

750 µM β-NAD                  0.1 M         0.075 ml

ddH2O                                      -             5.945ml

 

  • Making 0.1 M β-NAD: 0.25(g)/663.4(mw)/0.1(M)=3.768(ml). Add 3.768 ml ddH2O to 250 mg β-NAD.

  • Making 50 ml 1M (NH4)2SO4: 132.14(mw) x 0.05(ml) x 1(M)=6.607 (g). Add 50 ml ddH2O to 6.607 g (NH4)2SO4.

  • All the other buffers are from the Ambion Buffer Kit, 9010

Department of Microbiology
School of Medicine
University of Washington
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