topo ta cloning

Jose de las Heras jose.delasheras at virgin.net
Mon Dec 27 07:38:15 EST 2004


"D.K." <dk at no.email.thankstospam.net> wrote in message 
news:7pdvs0taumed0j7kgs1lkg32tia39cm9tc at 4ax.com...
> 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?
>
> I don't have an explanation but I've seen the same. Seesm to be 
> insert-specific?
> In some cases, over 90% are in one irionetation, in others - as expected,
> roughly 50/50.
>
>>2) After heatshocking the oneshot cells + ligation mix and adding SOC, how
>>long time (minimum) does the mix need to shake at 37 degrees? As it is AMP
>>selection I guess only around 10 min are needed, however the manual says 
>>one
>>hour? Could I go down to 10 min, and how will this affect the cloning
>>efficiency?
>
> 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.
> 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.
>
> DK

I once added ampicillin by mistake to the recovery medium, for an hour, then 
plated... and I got exactly zero colonies. Of course it could have been a 
bad transformation... but I doubted it as I had been doing transformations 
almost daily with the same cells without a hitch. I blamed the ampicillin, 
so I find your results interesting: you really tried back-to-back (ie: amp 
vs. no amp post transformation) and had good growth even with amp right 
after transformation? I wonder what went wrong in mine...

Jose

Jose 





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