
'lace-like texture' on my northern blot bands
#1
Posted 28 April 2011 - 11:02 AM
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.
#2
Posted 29 April 2011 - 09:45 AM
could you show us a picture?
talent does what it can
genius does what it must
i used to do what i got paid to do
#4
Posted 01 May 2011 - 03:52 PM
#5
Posted 02 May 2011 - 07:50 AM
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.
talent does what it can
genius does what it must
i used to do what i got paid to do
#6
Posted 06 May 2011 - 07:34 AM
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)
#7
Posted 06 May 2011 - 11:17 AM
if performed slowly and carefully then you should not get bubbles.Won't a rolling motion technique trap too many bubbles? (I'm using the roller on western blots and get good blots there)
i also use this method for westerns and have no trouble with trapped bubbles.
talent does what it can
genius does what it must
i used to do what i got paid to do
#8
Posted 06 May 2011 - 05:17 PM
#9
Posted 08 May 2011 - 11:14 AM
if performed slowly and carefully then you should not get bubbles.
Won't a rolling motion technique trap too many bubbles? (I'm using the roller on western blots and get good blots there)
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