2 replies to this topic

[phyros22]

i need help with this thanks in advance!

I am trying to PCR a protein that is found on a plasmid.. do i need to extract the plasmid from my organisms beforehand or can i just use a bead-beating DNA extraction and PCR on that?

[smu]

phyros22, on Dec 1 2009, 11:05 AM, said:

i need help with this thanks in advance!

I am trying to PCR a protein that is found on a plasmid.. do i need to extract the plasmid from my organisms beforehand or can i just use a bead-beating DNA extraction and PCR on that?

I think it depends on how well you want the PCR to work. You can do as you suggested, but it will be messier than if you were to purify the plasmid away from genomic DNA. But, if doing such a plasmid extraction is a lot of work then you might take the easier route.

[pDNA]

it also depends on the organism you are using ...you can easily do a colony PCR using E. coli, but you might get problems with other cells (e.g. gram+ that have a s-layer, yeasts).

So if you are using a organism with a tough membrane ...do a plasmid prep instead ...otherwise you will kill your Polymerase by a too long cell denaturing step in your PCR.

Regards,
p