Southern,DIG

Paul Lacoste lacostepaul at hotmail.com
Tue Jun 11 04:53:41 EST 2002


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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