Northern blot re-probing

ELADER at AMBION.COM ELADER at AMBION.COM
Mon Feb 23 17:15:40 EST 1998


In article <01bd3d56$38e73440$572a9b95 at pc0287.irad.bbsrc.ac.uk>,
  mullen at bbsrc.ac.uk wrote:
>
> 	I am wondering if anyone has had problems re-probing Northern blots. I
> initially probed my blot with a P-32 labelled PCR probe for a
> constitutively expressed gene (beta-actin) and was satisfied that my
> loading was OK. I had problems getting this control probe off the filter
> because it had dried! When I tried to reprobe the filter with another
> probe, I got lots of black spots of radioactivity on the filter with no
> bands (which I later found out to be due to Denhardt's solution which had
> expired).
>
> 	Having rectified this, I found upon subsequent re-probing that even
the
> control probe (beta-actin) did not work on my filter - most unusual! Has
> anyone got any ideas? The filter is Amersham Hybond-N (Nylon). I would
> really like to re-use the filters because of my small amounts of sample.
>

First, use a positively charged membrane (e.g. genescreen plus or hybond N+)
and crosslink with a stratalinker. Then use our strip ez kits to make probes
that come off the blot under mild conditions. Yo can reuse your blots 10X with
this system. Oh, hyb in a formamide buffer - it inhibits nucleases. Don't use
crude BSA in the blocker - full of RNase.

Eric
Strip EZ- www.ambion.com

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