We also have a Bio-dot apparatus available now, but the RNA seems not to stick to the membrane. I would be glad if you could share suggestions/experimental protocol for that
Which type of membrane are you using ? Nylon or Nitrocellulose ? Do you fix the RNA after blotting it on the membrane ?
Have a look on this article from Thermofisher
https://www.thermofisher.com/ch/en/home/references/ambion-tech-support/northern-analysis/tech-notes/membrane-transfer-and-crosslinking-for-rna.html
Thanks for your answer! I'm using nitrocellulose membrane and doing double UV crossing after spotting, as many protocol say. I have no problems with the visualisation, but the amount of RNA spotted doesn't seem exactly the same! (Maybe some pipetting error?) I also repeated RNA quantification but nothing changed
I wonder if you RNA is in good shape with a double crosslinking I doubt very much that you don't see your RNA might be a denaturation as ribonucleotides won't stick the same as intact RNA. Did you run your sample on a gel to visualize them ?
As I've written, I see RNA in dot blot, no problem with that. The only problem is equal loading of the RNA on the membrane in the different dots
Sorry for misreading then, are the samples different RNA or the same sample loaded several times ? If so is it from the same batch ? Which volume do you spot I experienced that sometimes with small volumes not everything is effectively spotted on the membrane it might just dry out on the side of the well of the dot blot apparatus
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