Mysterious probes do not bind southern

Harry Witchel Harry.Witchel at bristol.ac.uk
Wed Jul 26 06:22:54 EST 1995


I have a set of 10 hox-like sequences formed from a single PCR reaction 
from DNA of a ctenophore, Beroe ovata.  I have tried to Southern with 
these probes, and all but one of them has failed as far as I can tell.  
Although it is possible that my PCR rxn had DNA contamination from other 
sea critters in the gut of my ctenophore, I starved them before making the 
original DNA prep to be PCRed.  Before assuming that all my sequences 
(except for one) are rubbish, I want to double check what my blots might 
be up to.

Blotting failure takes one of two forms: either the probe fails to bind 
anything except for the lambda hind3 control (in which case I lower the 
temperature), or the probe binds as a smear to all DNA, in a pattern 
identical to what I see in ethidium (in which case I raise the 
temperature).  With each separate probe, I try a variety of temperatures 
and either get nothing or everything.  Have I overlooked any 
possibilities?  My protocols follow.

My original gels have a solid 10-20 ug  of digested DNA per lane (I run 1 
lane of lambda hind3, 3 different digests, and one lane of undigested DNA 
control), and the digestions seem to work; usually only one of three 
digests will have any visible repeat bands, and the lane of undigested DNA 
actually smears lightly down to 9 kb, but the DNA smears significantly 
lower when digested.

My transfers seems to work because lambda probes bind to the filters quite 
avidly.  I do not have a percentage-bound figure, but I use capillary 
transfer with 20X SSC, Zeta probe, and fixation by oven drying for one 
hour.

My probes seem to be well-labelled (10^9 dpm/ug).  I use 25 or so ng of 
cut out template crush eluted from acrylamide gels, and using DE81 paper 
my labelling runs between 30-80% incorporated counts, which is cleaned up 
in a Pharmacia Nick column to >90% incorporated, 10-20 million cpm on a 
32P setting, using a-dCTP that is less than 2 weeks old, 250 uCi/rxn, 25 
ul rxns, using an Amersham Multiprime kit with an overnight incubation.

The hybridizations also seem to work, again because lambda probes in the 
same tube bind to the standard lane.  Hybridizations are performed between 
 60-67 degrees, after 20 min of prehybridizations, in .25 M Na2HPO4 pH 7.2 
+ 7% SDS, in hybaid bottles in a rotisserie, 5-10 mls of fluid / tube.  
Before adding the probe it is prepared as follows: after Nick column the 
probe is in 300 ul TE, and I add 10-50 ul of the lambda probe and 50 ul of 
10 mg/ml sheared herring sperm DNA, the mixture is boiled 3 min, cooled on 
ice 3 min, squirted into 5 mls of hybridization buffer, and the buffer + 
probe are added to the tube with the pre-hybed blot.



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