Nonradioactive vs. radiolabelled probes
James N Petitte
jnppo at unity.ncsu.edu
Thu Mar 3 11:50:10 EST 1994
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.
J_Petitte at NCSU.EDU
More information about the Methods