Help! Genomic probe gives smears!
eatkinso at gpu.srv.ualberta.ca
Wed Feb 9 16:53:55 EST 1994
I hope this gets through. I'm using a new news reader that I'm not too familiar with and it's not as user-friendly as the last one I had access to. Anyways
here's the question:
In order to analyze a hologous recombination even in mouse ES cells, I need to
probe genomic Southern blots with a genomic probe derived from sequences
approx. 3 kb downstream from the last exon of our gene. I don't have
sequence info. for the region the probe comes from, but I do have a few RE
sites mapped. The piece of DNA I wanted to use for a probe is a 1700bp
Hind III fragment. Unfortunately, this ppiece of DNA just won't give me any
bands on my Southerns, only ugly smears. The DNA on the filters is good, though, since when I use a cDNA probe I get really nice looking bands. It's been
suggested that the most probable cause for this genomic probe not working is
that it contains repetetive elements. Seems reasonable. It has also been
suggested that I should try to cut the 1700 bp Hind frag into 2 or 3 pieces,
label each one of them, and see if a smaller fragment will work as a probe.
This also seems reasonable, but it will also take a bit of time. Does
anybody out there have any suggestions of how I might be able to use my originalprobe by somehow blocking the repetetive elements of the filter DNA? Would using sonicated mouse DNA as a blocking agent in my prehyb. work? I've never
heard of this, but somebody else has suggested it. At present, we don't
use any kind of DNA in our hyb or prehyb solutions for a blocker, just
ATP, pyrophosphate, and Denhardt's. It's never been a problem before.
I *know* I could go through all these possibilities myself, but if anybody'
has any experience with this kind of thing, I'd really appreciate it if'
you could pass it on.
Thanks in advance
Frustrated Ph.D student
U of Alberta
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