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plasmid standard curve qPCR

plasmid standard curve qPCR - Protocols and Methods Forum

plasmid standard curve qPCR - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 01-07-2009, 10:06 AM
Severin, Ina
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Default plasmid standard curve qPCR



Hi all,

I am currently working on the quantification of gene copy numbers and
transcripts and got to the point where I need to make my standard curve.
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? And in how far could the buffer used for the digest
interfere with my qPCR reaction? Do I need to clean my plasmid solution
(and if yes, what's the best way)?



Many thanks in advance!

Ina





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  #2  
Old 01-08-2009, 03:13 PM
Duncan Clark
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Default plasmid standard curve qPCR

Historians believe that in newspost
<[Only registered users see links. ].net > on Wed, 7 Jan 2009,
"Severin, Ina" <[Only registered users see links. ].nl> penned the following literary
masterpiece:

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


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.


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.
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