Striping and reprobing northern blots?

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Aug 16 10:24:34 EST 2001


Alan,

You may be hard pressed to find a treatment that will allow you to strip
and reprobe so many times.

I personally have used boiling 0.1X SSC, 0.1 % SDS to strip northerns a few
times without noticing much degradation in signal.  This treatment works
well for me if the probe is DNA.

I've never had great luck stipping RNA probes using any treatment like
this.  About the only thing I've found that works well for stripping RNA
probes is Ambion's StripEZ kit (no affiliation), which incorporates a
modified C into the probe that can be chemically broken down (with some
iodine conconction they provide, I think).  This does allow milder stipping
conditions, e.g. 60 degrees or so.

I recently noticed that they have a similar kit for DNA probes.  Although I
think this would be a waste of money for routine stripping and reprobing
(i.e. reprobing only once or twice) since a conventional boiling approach
works pretty well, in your case it might be worth it since you could use
gentler stipping conditions.

Hope this is helpful.

Mike


>Hello,
>
>I am looking for some advice on stripping and reprobing northern and
>southern blots.  I am trying to figure out the best method of stripping
>blots to maintain a good signal and still strip the majority of old probe
>from the blot.  I have been using boiling 0.1% SDS to strip blots for some
>time, but it seems to cause the northern blots to go bad after several
>treatments (The sensitivity seems to decrease greatly after 3 strips.  I
>know there are numerous ways to chemically strip the probe from the blot for
>example this works well for southerns (100mM NaOH, 10mM EDTA, 0.1% SDS) and
>neutralizing the blot in 5x SSPE.  Could anyone offer me some advice on how
>to strip northern blots very efficiently and gently?  I need to probe the
>same northern blot as many as 12 times before checking the control probe.
>Are there any solutions I should avoid for northern blots that are normally
>used with southern blots i.e. NaOH?
>
>
>
>Thanks
>
>Alan Smith
>
>---


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

---




More information about the Methods mailing list