Digoxigenin DIG Southern Blots
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Digoxigenin DIG Southern Blots
Does anyone out there have experience with using Boehringer-Mannheim's digoxigenin-labeled (DIG-labeled) oligonucleotide probes to detect target genomic DNA on their Southern blots. I am getting good transfer of the genomic DNA to a nylon membrane. I'm having some
problems getting enough of my probe DIG-labeled however. When I do the hybridization, I don't see any bands at all. The DIG-labeled molecular
weight marker ladder shows up fine on my blots however. Any suggestions
will be highly appreciated.
Bob Hummel
Newsgroup archive
First:
Check the efficiency and specific activity of your oligoprobe. Compare
a dot-blotted serial dilution with a standard from Boehringer.
Second:
Transfer a serial dilution of a plasmid containing the target sequence
from gel to a membran and prove the sensitivity of your
hybridization/detection-system. IMHO the sensitivity of your system is
much to low to detect a single copy gene in genomic DNA.
I would switch from oligo probe to a >300bp random primed cDNA probe,
hybridization and washing is easier to handle with a longer probe. In
my experience the sensitivity of your assay will be 10 to 100x
increased.
Hope this helps.
U. Maier
Newsgroup archive
Last update 07-Jul-2001, Rating Good of 7 votes.
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i am doing southern hybridization for copy number analysis with dig labieling and i am getting very good results with pcr probe of 1.5kb.
Rating: Excellent!
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,
I am using “DIG High Prime DNA labeling and Detection Starter Kit II” from “Roche”.
I labeled my probes (~ 450bp each), performed serial dilutions with the control DNA from the kit and spotted them on the membrane – everything worked just fine. Yet, when I did real experiment using wt digested genomic DNA I got whole line with the DNA smear labeled with no distinct bands whatsoever (I expected ~11 kb). The background of the membrane however was OK (not too high).
I used the commercial protocol:
Denaturation and neutralization of the gel prior to capillary transfer (20X SSC), UV crosslinking, air-dry, prehyb. at 420 for 2h, addition of denatured DIG labeled probe (5 min boiling), hybridization over night at 420, stringency washes, blocking, incubation with an antibody, washes and development.
I would really appreciate if somebody has any suggestion what could be a problem.
Thanks,
Niranjan
Rating: Good
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Hi. Does anybody have a recipe for prehyb buffer for DIG southern blot? Thanks.
Rating: n/a
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Hello,
I am using “DIG High Prime DNA labeling and Detection Starter Kit II” from “Roche”.
I labeled my probes (~ 450bp each), performed serial dilutions with the control DNA from the kit and spotted them on the membrane – everything worked just fine. Yet, when I did real experiment using wt digested genomic DNA I got whole line with the DNA smear labeled with no distinct bands whatsoever (I expected ~11 kb). The background of the membrane however was OK (not too high).
I used the commercial protocol:
Denaturation and neutralization of the gel prior to capillary transfer (20X SSC), UV crosslinking, air-dry, prehyb. at 420 for 2h, addition of denatured DIG labeled probe (5 min boiling), hybridization over night at 420, stringency washes, blocking, incubation with an antibody, washes and development.
I would really appreciate if somebody has any suggestion what could be a problem.
Thanks,
Alex
Rating: Good
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like Monali, i have tried southern blot using Roche high prime DNA labeling and detection starter kit II twice but no band detected on membran. what is the problem that make no band on the membran? i am waiting for suggestions. Tanks
Rating: Good
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I am using Roche high prime DNA labeling and detection starter kit II for southern hybridization.there is good transfer of DNA on nylon membrane and probe on testing lebeling efficeincy shows well labelled. But after hybridization there is no signal of band and showing darkback groundon X-ray film
Rating: n/a
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very technical information supplied by the website.
Rating: Excellent!
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We have used your DIG method for probe labelling ,g-DNA digestion is quite fine , our probe size is ~1kb ,but on the blot there is no signal , so is it the substrate or the probe concentration causing the problem ??
Monali
Rating: Good
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Hi, I am looking for a reliable method to determine gene copy number. I know it is possible to do it by Southern of pre-digested genomic DNA, but I don't know how good this method is. Any comments of suggestions?
Gonzalo
Rating: Good
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