Northern blot re-probing

Mon Feb 23 17:15:40 EST 1998

In article <01bd3d56$38e73440$572a9b95 at>,
  mullen at 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
> 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.

Strip EZ-

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