Turn heatblock on to 95C.
For samples in water or ethanol, dry down appropriate amount of RNA, and include a tube with 1ul of tRNA or Glycogen (Sigma), this is the negative control.
Each sample should have the following:
24ul Formamide
2ul 0.6M PIPES
2.4ul 5M NaCl
0.3ul 0.1M EDTA
2 x105 cpm main probe
5 x104 cpm loading control probe
DEPC water
Total Volume 30ul
Mix samples well. Heat @ 95C for 10 minutes. Incubate 4 hours @ 55C.
RNase Digestion Buffer:
300mM NaAc
10 mM TRIS
5 mM EDTA
To each sample add 350ul Digestion buffer.
Add 1ul of 4mg/ml RNase A and 0.4ul of 10u/ul RNase T1.
Incubate @ 30C for 45 minutes to 1 hour.
To each sample add 10ul of 20% SDS and 2.5ul of 10mg/ml Proteinase K. Incubate @37C for 15-20 minutes.
Extract once with 400ul of Phenol/Chloroform/Isoamyl Alcohol (25:24:1)
Transfer the supernatant to a new tube. Add 1000ul of 100% ETOH and 1ul of 10mg/ml of Glycogen. Mix well.
Incubate samples at -70C for 30 minutes or in a dry ice/ETOH bath for 10 minutes.
Spin in microfuge for 15 minutes. Aspirate ETOH. Allow pellets to air dry.
Resuspend in 8ul of Formamide based loading dye. Allow to sit at RT for 5-10 minutes, with frequent mixing.
Heat samples for 5 minutes @ 100C. Load onto a 5% polyacrylamide/7M Urea denaturing gel with 2000cpm of a molecular weight marker (dCTP labelled pBR 322 MspI digest works nicely).
Run gel @ 38-42 mA. Dry gel, expose to film O/N at -70C with an intensifying screen.
| Problem | Possible Solution |
|---|---|
| No Discrete Bands, Only Smears : | RNA degraded, or the the RNase Digestion was too harsh. Try reducing the concentration of RNase A to 1 mg/ml or digest for a shorter period of time. Check your RNA for condition. |
| Bands Too Large, High Molecular Weight Artifacts: | The RNase Digestion was inefficient and unable to effectively trim down the RNA:RNA duplexes. Try RNasing longer or increasing the RNase concentration. Incompletely linearized template DNA. |
| Bands in the Negative Control Lane: | Inefficent RNase Digestion (see above). Sense template contaminating riboprobe. Insufficent DNase digestion of riboprobe. |
| Many Lower Molecular Weight Bands Under the Main Band: | There can either be premature stop sites in the probe leading to smaller probe sizes, therefore smaller products. This can also stem from overdigestion by RNase A which will break-up the duplexes if the concentration or digestion time is too long. |
| No Signal At All: | You did generate an antisense probe right? |
[NextPage] The "Neverfail" Northern Blot Hybridization
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 65C.(optional)
2) Drain solution and replace with prehyb solution. Prehyb for at least 2 hours at 42C.
3) Denature the probe at 95C 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 42C.
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 55C.
10) Blot membrane dry with Kimwipe, cover with Saran Wrap and expose to film. 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.