Cloning problem

gmclab221 nayers at cctr.umkc.edu
Tue Mar 17 15:29:51 EST 1998


Hello,
I wanted to run by an observation/problem I have been having in cloning
some PCR generated cDNAs to see if anyone has any ideas what might be
going on.  I am trying to directionally clone my PCR products and after
I transform my ligation reactions I am getting really promising numbers
of experimental colonies vs. background vector colonies (5-10X above).
However, when I do mini-preps and restriction analysis, all of the DNA
is high molecular weight junk that appears to not be cutting. Very few
mini-preps will even cut and  those never contain inserts. My question
is basically what is is happening to give such deceptive transformation
results? And has anyone ever experienced this? I think my cDNAs are
realitively unstable and may form tertiary sturctures, however, I
wouldn't think this would lead to high transformation efficiencies. This
phenomenon does not appear to be vector specific either as I  get the
same results with a 3 kb, 6kb, or 10kb vector.
Any suggestions would be greatly appreciated.
Thanks,
Nancy
nayers at cctr.umkc.edu




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