Northerns AGAIN, sorry!
mediablitz at home.com
Mon Feb 18 13:25:24 EST 2002
....after transfer gently rinse the membrane in the transfer buffer and then
crosslink it ...you might want to try taking the NaOH out of the transfer
buffer too (it 's at a low  but it still might cut up a lot of your RNA )
...what sort of message are you probing for? how rare is it? what size is
it? Do you get a signal from an actin or GAPDH probe?
...from what you describe the protocol should work fine for a "normally"
expressed gene...if it's a rare message you might want to try reducing the
stringency of your washes.
"Jon" <dariothemonkey at yahoo.co.uk> wrote in message
news:a4r6r8$aon$1 at dux.dundee.ac.uk...
> I know this has been done to death, but there seem to be so many
> on the northern blotting theme that I'm not sure which bits of my
> cobbled-together protocol are actually right for me, as I'm not getting
> signal on the autorad. I'm using downwards transfer from a
> formaldehyde-agarose gel (total RNA) onto Hybond N+ membrane and am
> good transfer within 2-3 hours (assessed using a transilluminator as the
> samples have been heated with EtBr prior to electrophoresis; here, I can
> very clearly see the markers and sharp ribosomal bands). I rinse the gel
> briefly in 10X SSC and the transfer buffer is 2X SSC, 10mM NaOH.
> OK, fine so far, I think. But, following transfer: should I wash the
> membrane? Air-dry it? UV Cross-link it? Or all of these, and in what
> I'm using a non-isotopic DNA probe, labelled with fluorescein (Gene
> Hyb: I've been doing this in 5X SSC, 50% formamide, 5x Denhardt's, 0.5%
> 0.1mg/ml denatured herring sperm DNA, 42oC, overnight. Pre-hyb was about 1
> As a rule, is 42oC appropriate for DNA probes and 65oC for riboprobes? Or
> does that depend on the formamide content of the hyb buffer? Do I actually
> need formamide for DNA probes? And high MW "crowders" like dextran?
> I'm doing stringency washes as follows: 1. 2X SSC, 0.1% SDS,
> 0.1X SSC, 0.1% SDS
> - both at 42oC.
> Detection is by ECL using CDP-star.
> So many questions! If people are willing to share their experiences or
> suggestions, I would very much appreciate the help.
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