Prehybridization/Hybridization Solution

0.1% SDS
50% Formamide
5x SSC
50 mM NaPO4, pH 6.8
0.1% Sodium Pyrophosphate
5x Denhardt"s Solution
50 ug/ml Sheared Herring Sperm DNA

1) Air dry blot. Crosslink in Stratalinker or 30 seconds on UV Transilluminator. Wash the blot for 30 minutes in 1x SSC, 0.1% SDS at 65℃.(optional)

2) Drain solution and replace with prehyb solution. Prehyb for at least 2 hours at 42℃.

3) Denature the probe at 95℃ for 5 minutes.

4) Replace prehyb solution with fresh, preheated hyb solution containing 1 million cpm of probe per ml of hyb solution. (Average hyb is 10 million cpm total).

5) Hybridize overnight at 42℃.

6) Wash # 1: Fresh prehyb solution, 30 minutes at RT.

7) Wash #2 and #3: 2x SSC, 0.1%SDS 30 minutes each at RT.

8) Wash #4: 1x SSC, 0.1% SDS for 30 minutes at RT.

9) Wash #5: 0.2x SSC, 0.1% SDS for 45 minutes at 55℃.

10) Blot membrane dry with Kimwipe, cover with Saran Wrap and expose to film.

Troubleshooting and Tips

Excessive Background on film:	Speckled background indicates that there are unincorporated nucleotides in the probe. Blotchy backround indicates non-specific hybridization. This can be caused by salt still on the blot before the hyb. Be sure to wash the membrane. Can also indicate a lack of agitation during the hyb.
No signal:	a) Probe targets intronic sequences. b) Probe not denatured prior to hyb. c) Hyb conditions too stringent. d) Washing conditions too stringent.
Hybridization of Ribosomal RNA:	a) Too much total RNA has been blotted. Do not exceed 20ug of total RNA or try to use poly A+ instead. b) Probe is too sticky. Increase the stringency of the hyb conditions.
Smears:	The RNA has degraded. Stain the gel with Ethidium Bromide or SYBR Green II to ensure the RNA is intact. Can also stain filter with Methylene Blue to ensure the RNA did not degrade during transfer process.
Stripping blots for reprobing:	Add boiling 0.1% SDS, and wash for 30 minutes. Reprobe starting with step 2.