mbjpl at seqnet.dl.ac.uk mbjpl at seqnet.dl.ac.uk
Wed Sep 14 07:24:53 EST 1994

Dear all,

We are having trouble with Invitrogens Eukaryotic expression
vector pcDNA3.  The trouble started with the cloning of a rather
dirty PCR product which consisted of about four bands of about
equal intensity.  Normally we would not have persevered with such
a product but we are constrained to use the chosen  regions by
the gene we are investigating.  We cut the correct band (at least
the band of the correct size) from an LMP gel and cloned into a
TA clone (pCRII). We cleaved pCRII and recovered a product which
we attempted to clone into similarly cut pcDNA3.  This cloning
failed about three times despite reasonable quantities of DNA
apparently well cut vector.  Many amp resistant "monsters" were
obtained. We had checked the efficiency of the Bugs (Top10F')
and changed the restriction enzymes.  The transformation controls
looked OK.  Another strange phenomena was that two distinct sises
of colonies were seen and their ability to grow on the plate
correlated with the growth in liquid culture with the small
colonies taking about 24hrs to grow to stationary phase.  On the
fourth attempt a single positive colony was obtained from a small
colony.  This we grew to maxiprep stage although the yield was
low due to poor growth.  Using the DNA produced we were able to
transfect Cos7 cells and express the gene as measured by MAb
reactivity in an IPX assay.  Unfortunately the next stage of the
study utilizes site directed mutagenesis.  We hoped to use single
stranded DNA derived directly from the vector to do this
unfortunately ss-DNA refuses to be produced from this weakly
growing clone.  We seem unable to use helper phage M13K07 as the
vector appears to give Kanamycin resistance!  We thought that
maybe Neomycin resistance which the vector does have may be the
problem but have not heard this before.  Has anyone got any ideas
what is going on or have any advice.  Our present (rather
defeatist) strategy is to start again.  Clean up the PCR product
(if we can) and use a different expression vector.

Paul Lowings.                            mbjpl at uk.ac.dl.seqnet

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