ligation of blunt PCR products

Michael Szardenings msz at bio.embnet.se
Mon Jan 15 04:33:27 EST 1996


In article <4d1bf4$ojr at academ00.mty.itesm.mx>, calaniz
<calaniz at academ01.mty.itesm.mx> wrote:

> Do anybody knows if there is a method for efective blunt end ligation of 
> PCR products? please I need it for my master degree thesis in protein 
> engineering.
> Thanks
> 
> Cecilia Alaniz

Yes, this is how it works, in our hands:
1. Primers are kinased before the PCR reaction with the Ready-to-go kinase
from Pharmacia, the kit worked well for up to 500 pmol oligo per reaction
(add ATP to about 1mM!).
2. Use Vent polymerase for amplification and prepare fragment on a prep
gel or some silica.
3. ligate into 'whatever-you-want-dephos-or-not-vector', has all worked
well, but use a buffer with PEG or similiar. Best results I usually get
with BRL's 5xligation buffer or use (again) Pharmacias 'Ready-to-go'
ligase.

Regards
      Michael

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