8 replies to this topic

[Rhinovirus]

hi
I'm doing a northern blot and get these lace-like textures on my bands (practically all over the membrane).

I run the RNA on a formaldehyde gel in X1 MOPS buffer, than rinse it 2X15 min in X20 SSC.

transfer is done to a positively charged nylon membrane using the capillary method with X20 SSC for 4 hours. (needless to say, while stacking the 4 3mm whatman paper on top of the membrane I roll out all air bubbles).

Just after the transfer I can already see the 'texture' on the blue background (on the membrane area to which the blue loading buffer was transferred)

That 'texture' can be later seen on the bands I get.

I tried a different membrane with no difference - any ideas?

thanks.

[mdfenko]

is the texture anything like the texture of the whatman paper?

could you show us a picture?

[Rhinovirus]

The smear is on purpose, for the texture to be more visible but its just the same with clear bands.

Attached Thumbnails

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[bob1]

Salt crystallisation?

[mdfenko]

is this something new for you? what has changed since your last "good" blot?

have you tried a different lot of the whatman paper? the texture looks like it may be the pulp pattern of the paper.

instead of rolling out the paper to avoid air bubbles try laying the paper with a rolling motion (i don't know if i'm explaining it well, lay one edge on top and then let the rest down slowly). the roller may be breaking bubbles under the paper but not getting rid of them.

[Rhinovirus]

Salt crystallisation?

I thought of that but I'm using X20 SSC as described in most protocols - maybe I should try a bought solution instead of making it myself.

is this something new for you? what has changed since your last "good" blot?

have you tried a different lot of the whatman paper? the texture looks like it may be the pulp pattern of the paper.

instead of rolling out the paper to avoid air bubbles try laying the paper with a rolling motion (i don't know if i'm explaining it well, lay one edge on top and then let the rest down slowly). the roller may be breaking bubbles under the paper but not getting rid of them.

Since I started I never got a good blot.
I tried a different whatman and also tried adding a finer layer between the whatmans and the soaking paper - no difference.

Won't a rolling motion technique trap too many bubbles? (I'm using the roller on western blots and get good blots there)

[mdfenko]

Won't a rolling motion technique trap too many bubbles? (I'm using the roller on western blots and get good blots there)

if performed slowly and carefully then you should not get bubbles.

i also use this method for westerns and have no trouble with trapped bubbles.

[bob1]

I agree, I use the rolling the paper down technique and have never had a problem. You can still use a roller afterwards if you want to get a tighter layering.

[Rhinovirus]

Won't a rolling motion technique trap too many bubbles? (I'm using the roller on western blots and get good blots there)

if performed slowly and carefully then you should not get bubbles.

i also use this method for westerns and have no trouble with trapped bubbles.

I agree, I use the rolling the paper down technique and have never had a problem. You can still use a roller afterwards if you want to get a tighter layering.

Thanks,
I'll try it and see how it gows