Slot blot of PCR rxns?

Lauryl Maiya J Nutter lmjnutte at acs5.acs.ucalgary.ca
Mon Oct 30 18:04:11 EST 1995


Hi,

I am using PCR to screen for insertional mutations at a
particular locus in Drosophila.  My screening protocol consists
of parallel PCR reactions.  One is a positive control using two
GSPs giving a 168 bp product.  The other is a screening reaction
with four GSPs (all in the same direction) and a primer specific
for the conserved end of the transposon which is mobilized and
(hopefully) will be inserted into the locus near enough to one of
the GSPs to give a product.  Currently I load the PCR reactions
onto a 1.5% agarose gel for Southern analysis.  I probe with
random-primed DIG-labeled full-length cDNA, followed by
chemiluminescent detection.  The positive control reactions give
an appropriately sized band, indicating that the DNA is
amplifiable.  A band appearing in the screening lane indicates an
insert (I don't have one of these yet).

My question is can I do slot blots of the PCR reactions, without
any sort of cleanup, instead of Southern analysis?

I am simply looking for a yes/no answer in the initial screen.
Sizing of the product can wait until the next level of
screening.  Slot blotting would allow me to handle more samples
faster, since I would not have to do the overnight blotting step
or be limited to the current 40 samples/gel.  (I have tried using
downward blotting techniques, but find that with a 1.5% gel,
transfer is rarely complete, even after 5 hours.)  I am currently
doing the screen on 160 different DNA samples (ie. 320 PCR
rxns).  However, if I need to do phenol-chloroform extraction on
each reaction before slot blotting, then the potential for loss
of what may be a fairly low quantity PCR product outweighs the
time savings.

Has anyone tried direct slot blotting of PCR reactions?  With
chemiluminescent detection?

Thanks in advance for any help that you can give me.

Lauryl Nutter
Dept. Biological Sciences
University of Calgary
lmjnutte at acs.ucalgary.ca




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