Northerns AGAIN, sorry!

Jon dariothemonkey at yahoo.co.uk
Mon Feb 18 10:32:55 EST 2002


Hi,

I know this has been done to death, but there seem to be so many variations
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 any
signal on the autorad. I'm using downwards transfer from a
formaldehyde-agarose gel (total RNA) onto Hybond N+ membrane and am getting
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 order?

I'm using a non-isotopic DNA probe, labelled with fluorescein (Gene Images,
Amersham).
Hyb: I've been doing this in 5X SSC, 50% formamide, 5x Denhardt's, 0.5% SDS,
0.1mg/ml denatured herring sperm DNA, 42oC, overnight. Pre-hyb was about 1
hour.

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,          2.
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.
TIA,
Jon.





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