advice needed: my PCR fragment gives low yield when grown up! Why?

Trond Erik Vee Aune teva at online.no
Mon Oct 29 02:14:48 EST 2001



Mike Levin wrote:
> 
> Hi everyone -
> 
>    I'm looking for ideas on the following problem. A post-doc in my lab did
> PCR to clone a fragment (by homology) of a flatworm gene. He got one - it's
> the predicted size (about 200 bp), and he now wants to get it into a vector,
> grow it up, and sequence it to see if it's the right thing. So, he ligates
> it into pBluescript (a high copy number plasmid), transforms bacteria, and
> does a miniprep on all the colonies (18 of them). All the bacteria grow fine
> on Ampicillin-containing media. But here's the weird part: while the
> vector-only control gives a nice plasmid DNA yield (suggesting his miniprep
> procedure is fine), all of the insert-containing ones yield very little DNA.
> There's just barely enough to see that there's even an insert in there.

Can you say for sure that the insert is of the correct length? If not
the cells can be a contaminant. If it is the correct fragment then it
seems like the insert is somehow influencing your cells, either be
slowing growth or by inhibiting correct plasmid replication.

Was the optical density for the control (vector-only) the same as for
the other cultures before prepping? If not your insert is somehow toxic
to your cells. I assume the insert is cloned into the polylinker and not
somewhere else where it can be expressed ;)

If the density on the cultures was approximately the same, then your
insert somehow inhibits regular plasmid replication. How this can happen
is difficult to say...what kind of gene is this?

You can try with another vector, but I would then try one with a
different oriV than PBluescript just to test out if there's some
reaction between your insert and replication initiation.

Kind regards,
Trond Erik Vee Aune
http://kibt.chembio.ntnu.no/molgen/




More information about the Methods mailing list