Southern,DIG

Mathias Holpert mholper at gwdg.de
Thu Jun 13 06:53:48 EST 2002


Hi Paul,

I think your problem really is the sensitivity. The DIG-system is fine
when it comes to probing plasmid DNA- but not sensitive enough for
detection of a single copy gene. When I wanted to probe a single-copy gene
from Toxoplasma gondii (which should have a smaller genome size than your
plant), I used a radioactive probe. I don't like working with
radioactivity and usually avoid it, but when it comes to genomic DNA and
single copy genes, nothing can beat radioactivity.

Hope this helped

Mathias

Paul Lacoste schrieb:

> Hello everybody,
>
> I encounter problems with Southern for a single copy gene from a plant
> DNA : I use 20 ug of DNA,digested by EcoR1 or BamH1, under vacuum
> transfer (after the transfer,there's quite no more DNA on my gel).I
> use a nylon membrane (Hybond-N from Amersham). My probe is made from
> the plasmid used to introduce the GUS gene in the plant : it is made
> by PCR with DIG-System from Boehringer Mannheim and its length is
> about 500 pb.
>
> Prehybridization Buffer
> 5x SSC
> 0.1% N-lauroylsarcosine
> 0.02 % SDS
> 1% Blocking Reagent
>
> Hybridization Buffer: same composition + 50 ng probe/ml solution
>
> Prehyb and Hyb tried at 68°C and 60°C. O/N Hyb
> Washes : 2x5 min with 2x SSC (0.1%SDS)
>          2x15 min with 0.2x SSC (0.1%SDS)
>
> Colorimetric Detection (NBT/X-phosphate): even O/N
>
> Results : no background, which seems surprising
>           the positive control,with the plasmid used to make the
> probe, works
>           no bands for the plant DNA,digested or not,which should be
> positive (tested by PCR)
>
> I think that the problem comes from the sensibility of the
> colorimetric detection and maybe from the membrane too (an Hybond-N+
> could be better).But I would like to have your advices (specially if
> you had the same problems in the same conditions) before buying a
> chemiluminescence kit.
>
> Thank you all in advance.
>
> Paul Lacoste




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