Striping and reprobing northern blots?

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Thu Aug 16 16:12:51 EST 2001


In <JIEHICBJDMGFLNGJIBDMIEFICEAA.alansmith at students.wisc.edu>, 
Alan Smith wrote:
>
>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?

You would definitely want to  _avoid_ using NaOH to strip! That would 
hydrolyze the RNA.

FWIW, I also use boiling 0.1% SDS (sometimes with 0.01 X SSC) to strip 
Northerns.

Honestly, I think you are pushing your luck in trying to get a dozen 
reprobings out of a Northern, no matter how you strip it. 

Perhaps you can combine some probes that hybridize to different-sized 
messages? For example, you should be able to simultaneously detect a 
5 kb, a 2 kb, and a <1 kb message on the same blot at the same time if 
their signals are not too extremely different. That might reduce the number 
of probings/restripings. Can you possibly run a duplicate blot or two? If 
you combine that and simuiltaneous probing, you might be able to reduce 
the number of reprobings to three or four.

Alternatively, design some RNase protection probes that would give 12 
different sized protections and use a sequencing gel to separate out the 
fragments.

Nick

-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu

 




More information about the Methods mailing list