Nonradioactive vs. radiolabelled probes

[James N Petitte]

Marion Freistadt (mfreis at lsumc.edu) wrote:
: Has anyone compared the sensitivity of non vs. radiolabelled probes in
: southern blots, all else being equal?  We are having difficulty detecting a
: single copy sequence used the Genius kit; but we know the gene is there by
: PCR.  The positive control in the southern (plasmid DNA) works, although we
: have not yet titrated the sensitivity, hoping it wouldn't be necessary.  I
: was thinking maybe I should go back to 32P.  Comments?


You should check out the paper in Analytical Biochemistry 210:235-244 
(1993), "Reduction of Background Problems in Nonradioactive Northern and 
Southern Blot Analyses Enables Higher Sensitivity Than (32)P-Based 
Hybridizations", by Blum, Meier, Frank, and Muller.

They describe a series of modifications to the Genius procedures that 
increase the sensitivity of dig-oligos and cDNA probes.  We are currently 
using their procedures for DNA fingerprints and have obtained the best 
autorads of any nonradioactive procedure we tried.  The only change we have 
made to their protocol is that we are using 1.5 M NaCl in 
washing buffer 1 rather than 3 M NaCl since we could not get all of the 
NaCl dissolved in the maleic acid buffer.  (I should ask them about 
that).  We have not tried it yet with a single copy gene but Fig. 5 
describes single copy detection with a 4 hour exposure.

Please let me know if it works in your hands.


James Petitte
NCSU
J_Petitte at NCSU.EDU