Protocol

  Denaturation

 1.   Prepare the following:

   25 µg total RNA/ 1µg poly A+ RNA                                      x      µl

   1 µg oligo dT19V (2 µg/µl)                                                      0.5   µl

   8 µg oligo dT30 (8µg/µl)                                                          1.0   µl

   GFP poly A+ RNA (10ng/µl)                                                  0.5   µl

   ddH2O                                                                                     x      µl

 Final volume                                                                           10.5   µl

 2.     Heat at 70 °C for 10 minutes, cool to 42 °C, slowly.

 Note: Heat in 70 °C heat block then take out block and allow to cool on bench top until temp is 42 °C.

  Reverse Transcription

 3.     To sample from above add the following:

 dNTP mix (800 µM dATP/dGTP/dTTP, 5 µM dCTP)           1.0   µl

 5x First Strand Buffer                                                              5.0   µl

 10x DTT                                                                                 2.5   µl

 [α-32P]-dCTP (10µCi/µl)                                                      5.0   µl

 SuperScript II (200 U/µl)                                                        1.0   µl

 Final volume                                                                           25.0   µl

 4.     Incubate at 42 °C for 1 hour.

  Hydrolysis of RNA

 5.     Sequentially add the following:

 1% SDS                                                                                 1.0   µl

 500 mM EDTA                                                                      1.0   µl

 2 N NaOH                                                                              3.0   µl

 6.     Heat at 65 °C for 5 minutes

 7.     Then add the following:

 1 M Tris.HCl pH 7.6                                                             10.0   µl

 2 N HCl                                                                                  3.0   µl

 8.     Incubate at RT for 10 minutes.

 9.     Add ddH2O                                                                      7.0   µl

 Final volume                                                                          50.0   µl

 

 10. Purify probe over G-50 Micro Column (Pre-spin col umn at 3500 rpm for 1 minute, apply probe to column, spin at 3500 rpm for 2 minutes.)

 Note: Save 1µl for counting and 1µl for denaturing gel.

  Prehyb membrane

 11. Incubate filter at 65 °C for 6-20 hrs in 10 ml Hybridization solution (5x SSC, 5x Denhardt�s, 0.5% SDS, 100 µg/ml ssDNA)

 

                                        5ml    10ml  15ml  20ml  25ml  30ml  50ml

 20x SSC                         1.25   2.50   3.75   5.00   6.25   7.50   12.5

 50x Denhardt�s              0.50   1.00   1.50   2.00   2.50   3.00   5.00

 20% SDS                        0.13   0.25   0.38   0.50   0.63   0.75   1.25

 ssDNA                           0.05   0.10   0.15   0.20   0.25   0.30   0.50

 ddH2O                           3.10   6.20   9.20   12.3   15.4   18.5   30.8

  Probe preparation

 12. Boil probe 5 minutes, place on ice at least 5 minutes.

 13. Add 2 µg poly A72 to 2 ml Hybridization solution.

 14. Add denatured probe to the 2 ml Hybridization solution.

 15. Incubate 1 hr at 65 °C.

 16. Add 3 ml Hybridization solution.

  Hyb membrane

 17. Incubate filter in 5 ml Hybridization solution for 20 hrs at 65 °C.

  Washes

 18. Wash membrane 5 minutes at RT in 2x SSC/0.1% SDS.

 19. Wash membrane 20 minutes at 65 °C in 2x SSC/0.1% SDS.

 20. Wash membrane 1 hour at 65 °C in 0.1x SSC/0.1% SDS.

 21 (Optional). If needed, repeat 1 hour at 65 °C 0.1x SSC/0.1% SDS.

 Adapted from Huntsman Cancer Institute & Katze Lab Protocols

Department of Microbiology
School of Medicine
University of Washington
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