Cloning with TOPO TA Cloning Kit

Aric Wiest awiest at ppserver.tamu.edu
Wed Nov 15 18:08:40 EST 2000


Hi Lukas,

Is your insert straight from the pcr reaction or do you gel isolate it?
I have used this kit many times with great success.



Aric

lukasfenner at hotmail.com wrote:

> Hi all:
>
> Who has experience with the TOPO TA Cloning Kit from Invitrogen?
> Control reaction with pUC18 plasmid (1ng) gives plenty of colonies
> (LB-Amp plate).
> With the pCR2.1-TOPO plasmid only (10ng) we get about 32 colonies
> (LB-Kana plate).
> Different experiments with/without additional salt solution, different
> amount of PCR products, additional Taq-Polymerase reaction before using
> did not give better results.
> The kit is at least six months old, stored at -20, competent cells
> (TOP10 one shot cells) at -80.
>
> From your experience, do you think the vector is gone bad?
> Do you know a better PCR cloning system?
>
> We use the LightCycler real time PCR from Roche with Fast SYBR Hot Start
> (Taq-Polymerase).
>
> Cheers,
> Lukas Fenner
>
> Sent via Deja.com http://www.deja.com/
> Before you buy.
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