Reagents:

20X SSC (3M NaCl, 0.3M NaCitrate, pH 7.0):

  1000ml 2000ml
NaCl  175g 350g
Na3citrate.2H2O 88g 176g

 - adjust pH to 7.0 with 1M HCl

 

100X Denhardt:

  100ml  500ml
Ficoll-400 2g 10g
Polyvinyl-pyrrolidone 2g 10g
Bovine Serum Albumin 2g 10g
ddH2O to 100ml to 500ml

 - filter sterilize

 - store at �20 °C in 25ml aliquots

 

Hyb Solution (5X SSC, 5X Denhardt, 50% Formamid, 1% SDS)

  5ml 10ml 15ml 20ml
20X SSC 1.25ml 2.5ml 3.75ml 5.0ml
100X Denhardt 0.25ml 0.5ml 0.75ml 1.0ml
di-Formamid 2.50ml 5.0ml 7.50ml 10.0ml
10% SDS 0.50ml 1.0ml 1.50ml 2.0ml
ssDNA (11mg/ml) 50ml 100ml 150ml 200ml
ddH2O 0.45ml 0.9ml 1.35ml 1.8ml

 

 

Protocol:

  1. Rinse gel with ddH20, 3X.
  2. Soak in 20X SSC for 45 minutes.
  3. Take photograph of gel.
  4. Soak nylon membrane -cut to size- in ddH2O for 5 minutes.
  5. Put sponge in container, fill with 20X SSC halfway up sponge.
  6. Put 3 20X SSC-soaked GB002-sheets on top of sponge.
  7. Place gel on top, remove bubbles.
  8. Cover with nylon membrane, remove bubbles.
  9. Successively add 1 GB002, 4 GB003 and 4cm GB004.
  10. Cover with glass plate and 0.4kg weight.
  11. Transfer overnight.
  12. Take structure apart, mark wells on membrane.
  13. Visualize and mark rRNA bands and RNA MW ladder on membrane.
  14. Rinse membrane in 2X SSC.
  15. Dry on GB003 and wrap in plastic foil
  16. UV cross-link (�Autolink� on Stratalinker).
  17. Take photograph of flattened gel to assess transfer efficiency.
  18. Pre-hybridize membrane for 4-20 hours in 5-10 ml Formamid (Pre-) Hybridization solution (FPH) at 42 °C.
  19. Boil 100µl probe for 10 minutes (use 1 x 106 cpm/ml FPH).
  20. Cool on ice, spin.
  21. Add to hybridization bag.
  22. Incubate 20 hours at 42 °C.
  23. Rinse in 2X SSC/0.1% SDS at RT, 3X.
  24. Wash in 0.2X SSC/0.1% SDS for 15 minutes at 42 °C, 2X.
  25. Wash in 0.1X SSC/0.1% SDS for 15 minutes at 65 °C, 2X.
  26. Rinse in 2X SSC.
  27. Wrap in plastic foil.
  28. Expose to phosphoscreen.

 

Notes:

  • After overnight exposure 5pg RNA can be detected with a probe labeled to a specific activity of 109 dpm/mg.

  • Probes labeled to ≥5 x 108 dpm/µg should detect transcripts that represent 0.01% of mRNA population with a blot of 10 µg total RNA or 0.0002% of mRNA population with a blot of 3 µg poly(A)+ RNA.

  • For stripping poor boiling 0.05% SDS on membrane, incubate for 10 minutes, repeat up to 3X. Rinse with 2X SSC.

Department of Microbiology
School of Medicine
University of Washington
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