problem with DIG Sourtherns

Daniel.Isenegger at nre.vic.gov.au Daniel.Isenegger at nre.vic.gov.au
Tue May 29 00:24:44 EST 2001



I have a problem with DIG labelled Southerns for npt II transgene detection
using the Boehringer users guide and kit.
I've used 10 ug of genomic DNA digested with appropriate restriction enzymes and
transfer to nylon membrane (N+) was ok.

Detection was done in clean plastic trays and 2 substrates were tested:
1) with CSPD I got high uniform background and low sensitivity of fragment
signal.  I could only just see the postive control and could not see the Lambda
marker (20 ng loaded of BM DIG labelled).  I'm using the standard hyb buffer at
68 C and prehyb and hyb conducted in roller tubes.  Stringency washes (at 68 C)
are also done in roller tubes.  The probe has been checked and is labelled ok.

2) with CDP-star my signal intensity improved considerably with the positive
control but still could not see Lambda markers.  The backgound was uneven with
clear and dark grey blotches.


If anyone has had similar problems and/or  have suggestions or tips to improving
the detection and background please let me know. Many thanks.
Daniel


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