Dear all,

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed May 19 09:25:29 EST 1993


kraj at crc.ac.uk (Mr. K.R. Raj) writes:

>I am in a bit of "fix" with my Southern blot. I am able to detect episomal 
>DNA of 8 kb in size on my Southern blot but am unable to detect it when I 
>digest the DNA with Bam HI which cuts the 8Kb DNA only once. I have tried 
>different batches of enzyme and buffers and several different DNA extracts.
>What I want to know is, if it is easier to detect supercoil DNA on Southern
>than linear DNA....does it make any sense?...if not what would possibily accoun
>account for the strange observation I observe with my experiments above?
>Any suggestion is appreciated.
>
>Ken Raj
>Dept of Pathology
>Cambridge.

It appears from your posting that you've left out a depurination step in your
Southern transfer. Is this right? If so, the coiled DNA has tranferred well,
but the linear DNA has not. Try a longer depurination or leave the gel on a UV
transilluminator for 10 minutes exposure before the transfer.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
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