Does phenol affect Norhern blot? - need your advice (Jul/25/2004 )
Now I am doing Northern blot with DIG-labeled probes. But this time, no signal appeared after the NBT, BCIP detection. I found there was too much phenol in total RNA. But I don't know if it is the reason. Anyone can give some advise, please?
Did you do any kind of stain on the northern membrane defore you probed it? If you had too much phenol in you RNA then it could be a problem with your transfer onto the membrane. However, you should be able to get rid fo the phenol by ethanol precipitating the RNA prior to running it on the gel to probe.
there are several stains you can use on a northern membrane but i normally use a methylene blue stain:
Methylene Blue stain:
0.03% methylene blue
0.3M sodium acetate (pH 5.2)
Stain membrane for about 1-2 minutes (if stain is new – NO LONGER - don’t stain for too long if the stain is new or you’ll never get it out!). you should see bands appearing on the membrane – if not, you may need to destain it slightly because it may be too dark). You will see 28S and 18S rRNA bands (28S on top and 18S on bottom) if you work with animal RNA - not sure about other types of RNA. Smearing down the lanes will indicate RNA degradation. Bit of smearing is ok but too much is not. This will tell you how well your transfer worked. Dont let the stain dry out too much on your membrane before you destain or it may be hard to get out. You can leave the membrane soaking in water in the meantime (but not for hours and hours!).
You will need to destain before probing the membrane. The stain can be reused heaps of times (the older it gets the longer you may need to stain). You destain the membrane by successive washes in 0.1X SSPE
0.1% SDS (add SDS after the SSPE and water or it will come out of solution in the high salt content of the SSPE). Keep destaining until the membrane is clean again. Then rinse in water and dry the membrane or probe it straight away.
This should tell you whether the problem was the phenol in your RNA because if it was, then the transfer would not have worked very well. Another important thing to know is that you should never phenol extract a DIG-labelled probe after you make it. This is because the DIG will separate into the phenol phase and you wont be left with anything!
hope all this helps!
Hi, Dear ros
Thanks for your advice!
I didn't do any stain after the transfer. Later, I will have a try!
You told me that a DIG-labeled probe can not be extracted by phenol. But my probe was prepared by PCR. Can it be used for Northers directly? I am afraid it will result in the degradation of the total RNAs.
i dont think that the DIG-labelled probe will degrade the RNA on your membrane. Once it is immobilised and bout to the membrane it should be ok for a while. When i do northerns, i don't always use RNAse-free reagents (eg. hybridisation buffers) with my membranes and they work well anyway. so i wouldnt worry too much about the DIG probe if it is synthesised by PCR.