improve the signal in Northern ?
crawford at comb1.comb.umd.edu
Fri Feb 26 09:44:00 EST 1993
In article <9302260344.AA11725 at fraser.sfu.ca>, peijunz at sfu.ca writes...
>I have a trouble in my Northern blot. The total RNA loaded was 20 ug
>and the signal still weak in my Northern. The longer time exposured to
>the film, the heavier background I got. How can I improve the signal
>as well as cut down the background?
>Thank you for your help.
>peijun_zhang_a at sfu.ca
I do a LOT of northerns (ahhh, the life of a grad student.....) and have
had mucho success doing the following:
- formaldehyde gels (final conc. of formaldehyde ~0.74M)
- run THIN gels, eg. 3-4mm. I use FMC SeaKem Gold for high
resolution and gel strength.
- use enough 1X MOPS running buffer so that the buffer is only
1mm above the gel
But probably, IMHO, the most important parameters re. poor signal and
high background are method of transfer, transfer to what, and pre-hyb/hyb
solution. My choices are:
* Transfer: Stratgene's PosiBlot
With no reservations at all this is superior
to any thing I havever tried AND quick too,
eg. complete transfer of RNA from a 3mm gel
in 30-45 minutes.
* Membrane: Strategene's Duralon-UV
I've tried a lot of 'em (nylon membranes from BioRad, S&S,
BRL, Amersham, etc. etc.) but at our institution
we get the best deal ($) from Stragene and the signal
is comparable (if not better) to more expensive membranes.
Also, forget baking, UV crosslinking is the way to go.
And...it can stripped over and over...
* Pre-hyb/hyb solutions: Stratagene's QuikHyb
VERY QUICK!! VERY LOW (read no) BACKGROUND! Pre-hyb
for 15 min, hyb for 1 hour, washing takes ~1.5 hours.
In my hands I have NEVER had a problem with signal/noise
ratio using this product.
I know this whole thing sounds like an advertisement for Stratgene. I am
in no way connected to Stratgene and I do not want to go to La Jolla for
a post-doc. I checked out a lot of different systems/gadgets and they just
outperformed the rest. Not all their stuff is good, I had tremendous
difficulties with their VAGE (vertical agaros electrophoresisis) system.
Using the above mentioned hardware I can detect very low levels of RNA
and get from whole cells to an autorad in 2-3 days. If anyone wants more
details, protocols etc please E-mail me and I'd be happy to share.
Like I mentioned, I do a LOT of northerns and have worked out a system
with a relatively high data/grunt work ratio, ie. lots of data with
little wasted time doing unecessary "grunt-aargh-?!@#@#$" work.
Please ignore any mistyping/spelling errors, lots of line noise today...
Eric Crawford | crawford at comb1.comb.umd.edu
University of Maryland | Voice: (410) 706-5664
School of Medicine | FAX: (410) 706-8162
10 South Pine Street | "standing on the shoulders of giants
Baltimore, MD 21201 USA | makes me tall" =(8-}
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