topo ta cloning
hzhen at freeuk.com
Wed Dec 29 15:17:09 EST 2004
> On Sun, 26 Dec 2004 22:46:55 +0100, "as" <nospam at spam.dk> wrote:
> >Hi. I have a few basic questions regarding topo TA cloning
> >1) When I TA clone a taq produced PCR fragment into a given TA topo vector
> >and subsequently sequence or colony screen the clones I usually find that
> >most of the clones are in the same direction? But there should be random
> >orientation of the fragments resulting in a 50/50 distribution?
The reason is usually toxicity of the gene product.
> If it's Amp then no recovery is necessary! It WILL happen on dishes. You
> migth recall that Amp does not kill cells - only prevents them from dividing.
Ampicillin is bactericidal - it causes cell lysis when the cell tries
to expand and divide. So it is only bacteristatic when the cell is at
stationary phase. Some may survive as spheroplasts or filaments
(especially if the concentration of the antibiotic is not high), but
they are unstable.
However you are right in saying the recovery step is unnecessary, I
normally don't bother. The cell wall is constantly being broken down
and rebuild in E. coli when it is growing, but the effect of the
antibiotic takes time to completely destabilise the cell wall and
cause cell lysis, and in that time, cells containing the
beta-lactamase gene would have time to synthesise the protein to
counteract the antibiotic.
> So, they will sit still on dishes for ~ one hour to synthesize enough
> lactamase and then will go on to divide. I've done tests back to back
> and the difference, where it exists, is marginal and can 100% be accounted
> for by the cell division without antibiotic during the recovery time.
> Of course, with kanamycin and the other stuff that poisons cells,
> the recovery is a must.
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