I need help on Ligations

R Daines rdaines at aol.com
Tue Apr 11 18:24:32 EST 1995

Define what your transformation control is please?  If is the pMON cut
with NCOI and EcoRI, then why should it ligate.  Most kits come with pUC18
or 19 to use as a transformation control.  Is this your control?  Also,
how far from the ends of your PCR fragment are your introduced restriction
sites?  Some enzymes require a hefty leader (endonucleases) while others
only require a few base pairs.  I don't recall the requirements of the
enzymes that you are using.  PCR generated DNA can also contain
transcription errors (probably not your problem).  And lastly,  triple
check your sequence to make sure you designed your primers and restriction
sites correctly.

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