topo ta cloning

as nospam at nospam.com
Thu Dec 30 04:23:29 EST 2004


Hi again.
Thanks for the answers.
I decided to test the difference between recovery and no recovery.
So I transfected 50 µl of invitrogen top10 cells with 1µl of ligation mix,
and after heatshock I plated 25µl on amp plates and the other 25µl I
incubated with SOC at 37 for 40 min.
On the first plate I got around 30 colonies and on the other plate, around
300. So I guess it is worth doing after all!
Happy New year.
AS

"as" <nospam at spam.dk> wrote in message
news:njGzd.640$gz5.450 at news.get2net.dk...
> 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?
>
> 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?
> Thanks
> AS
> DK
>
>





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