Cloning with TOPO TA Cloning Kit

lukasfenner at hotmail.com lukasfenner at hotmail.com
Tue Nov 7 05:25:54 EST 2000


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


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