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RNA transfer to nitrocellulose membrane (Northern blot)

RNA capillary transfer nitrocellulose

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#1 pailinyk

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Posted 08 March 2012 - 08:57 AM

Good morning everybody,
I have some problems with Northern blot. After overnight transfer of RNA from formaldehyde-containing agarose gel to the nitrocellulose membrane I always observe a "blind" spot at the middle of membrane with bromfenol blue lanes at the edges. At the gel its vice versa: blue spot at the middle and "empty" edges. It means that the transfer of RNA samples loaded to the central gel pockets is not so effective as for "side" samples (it was confirmed both by DIG-labeling system and using of radiolabeled probes: samples at the edges of membrane looked much brighter than samples loaded to the 3-4 pockets located at the middle of gel). I use upward capillary transfer with NaCl/NaOH-containing transfer buffer, stack of paper towels and 400g weight above it.
Can anybody please help me out and give any advice in order to fix this problem? Thank you very much in advance.

Respectfully,
Philipp

#2 bob1

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Posted 08 March 2012 - 11:50 AM

It sounds to me like your "salt bridge" hasn't been properly put together, or that the stack you are building is too short to wick effectively, or that your stack is wicking directly from the bridge (called short circuiting).

For the salt bridge, it needs to only be a couple of centimetres higher than the buffer level.

If the stack of paper towels is totally wet at the end of the transfer, it is too short - just add some more. I think I was using about 10 cm of paper towels.

For the short circuiting - place a thin strip of parafilm or saran wrap under the top and bottom edges of the gel to act as a barrier between the salt bridge and the paper towels, and ensure that your paper towels are not touching the salt bridge at any point and won't touch it when they start to get wet - cutting them to the same size as the gel will ensure this.

#3 pailinyk

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Posted 08 March 2012 - 12:13 PM

Thank you very much, Bob1, we'll certainly try it. Hope it will help us.

Philipp





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