UNEXPECTED TRANSFECTION RESULTS USING LINEAR PLASMID

paul gold pgold at MO.NET
Wed Sep 17 16:59:58 EST 1997


Dear Netters,

Recently I transfected a TK negative fibroblast cell line with a
linearized plasmid construct.  After selecting in HAT media we obtained
plenty of stables.  I performed Southern Analysis to determine the
extent of incorporation of our plasmid into the genomic DNA.  The
Southern Analysis gave a very nice pattern from our TK probe.  With the
TK probe some of the cells clearly had tandem repeats and some appeared
to be single copy.  Upon further analysis of the single copy
transfectants we were surprised to see that there appears to be no
plasmid DNA flanking region (including our gene) either 5' or 3' to the
TK gene.  All controls such as probe control and efficient DNA transfer
check out. My Question: Should we have used supercoil DNA?  Regards.

Paul Gold



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