I am not able to get the point Why do we use linear DNA after digestion of plasmid ? Why can't we use circular DNA to transform Pichia cells ?
Hi there,
Linearization of the transforming plasmid has 2 main advantages:
1) It improves the integration efficiency
2) It targets the doublehomologous recombination event near plasmid ends and therefore the site of integration within the genome is known
As Dominique said, you get more recombinants when intergration by replacement is sought. On the other hand, you could as well transform P. pastoris with the circular form of the plasmid. In this case you would get transformants in which the complete plasmid integrates at specific genomic sites (AOX or HIS genes) via a single-point recombination.
Also, when you perform your transformation with circular DNA you need at least 10 times more DNA (100 ug) than when you use linear DNA (approx. 10 ug), due to the efficiency is lower.
What is the percentage of cells undergoing homologous recombination in pichia cells transformed with linearised recombinant plasmid?
do we need to dephosphorylate the linearized vector before transforming them in to pichia?
- Badges
- Science topic