1 reply to this topic

[haui]

Hi,

Ihave to make my Northerns with riboprobe because my normal DNA sonde doesnt work correctly.
Who has experience with radioactive Probes with the Ambion Strip EZ DNA Kit. I want to use ATP. I heard ATP is maybe not so good???

I have clone my genesonde in SK- Bluescript. There I have T3 and T7 Promoter for the RNA Polymerases. Than I buy the T7 and T3 RNA Polymerase from Fermentas#EPO111.

Are there any protocols to make it so or what person has made before in such a way.

thanks for help.

[kajevr]

For Northern blots, I always used labeled UTP.  Not sure if there is any problem with using ATP.  I have not used the Ambion kit

If you have a plasmid with a T7T3 promoter, it is fairly easy to make probes.  I'm sure you can find a protocol somewhere..here's a basic description.

- Clone gene into T7/T3 vector
- Decide whether to use T7 or T3 for transcription (based on orientation)
- Linearize T7T3 plasmid (choose a restriction enzyme that cuts near where you want the transcript to end)
- Use linear plasmid in in vitro transcription reaction with labeled rNTP (make sure you add a  - small amount of the same cold rNTP used for labeling..helps with yield)
- Purify transcript
- Add to blot

Hope that helps!