Cloning LightCycler PCR products

Susanne Rohrer "srohrer(removethis)" at immv.unizh.ch
Wed Nov 22 03:21:02 EST 2000



lukasfenner at hotmail.com wrote:

> Does anyone has experience with cloning LightCycler PCR products? We
> have LightCycler real time PCR from Roche with HotStart Taq-Polymerase.

May I ask you why you use this machine for cloning purposes?

>
> For cloning we use the TOPO TA Cloning Kit from Invitrogen or the
> PCR-Script Amp Cloning Kit from Stratagene?
>
> Do you gel isolate it?
> Do you add an additional cycling with ThermoCycler (PE) after the
> LightCycler?
> Do you use special purification steps?
> Or do you only use ThermoCycler PCR products?

Theoretically, It should yield the same quality of product. The yield
should be enough for cloning from a 20ul reaction. I would purify it over
a spin column system to remove dye, primers, and dNTPs. if the product is
pure, then it should be fine. The only problem might be that Taq is not
proofreading. Any of the kits should work fine, although a kit is not
really necessary for cloning.

Susanne






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