plasmid standard curve qPCR

[Duncan Clark]

Historians believe that in newspost 
<mailman.1334.1231348039.29717.methods from net.bio.net> on Wed, 7 Jan 2009, 
"Severin, Ina" <I.Severin from nioo.knaw.nl> penned the following literary 
masterpiece:
>I have already isolated and linearized the plasmids containing the
>insert of interest but am (already for a while) wondering for what exact
>reason the linearization needs to be done. Unfortunately, I did not find
>much information in the literature (at least not in ecological
>research). Do the different formations of a plasmid behave differently
>in a qPCR reaction?

Supercoiled plasmid is not ideal as it can be refractory to 
denaturation. Therefore one plays safe with linearisation.

>And in how far could the buffer used for the digest
>interfere with my qPCR reaction?

1ug of plasmid of 4kb will be 2.32 x 10E11 copies. A 10,000 fold diln of 
that will have diluted out any possible buffer interference.

>Do I need to clean my plasmid solution
>(and if yes, what's the best way)?

Anyway you want but it's probably not worth it.

For more detail you can ask on the qpcrlistserv on Yahoogroups.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.