Plasmid as standard - Plasmid calculation (Mar/30/2009 )

Hello,

I am working on setting up a SYBR green assay to amplify a 113bp product from cDNA from virus. I have finished cloning this same fragment into a TOPO TA vector and have done a plasmid preparation for getting high yield of the plasmid.

I was wondering if anyone knows the calculation for finding out how many copies of the plasmid is present in the tube and how to go about diluting the plasmids. so as to get known copies to be used as a standard.


thekid.

thekid on Mar 30 2009, 01:24 PM said:

http://www.uri.edu/research/gsc/resources/cndna.html

very helpful.

thekid on Mar 30 2009, 02:24 PM said:

You are creating an internal problem.
The way you are going about it is flawed. In this way you will know accurately how many copies of plasmid you start of with but not what your titer was.
The problem is that the efficiency of the Real time reaction (or any other PCR for that matter) is different for plasmid/PCR product/cDNA.

I used to word at a pharmaceutical company that worked on Hepatitis C virus (witch is a RNA virus). In order to calculate the titer in blood samples we extracted the RNA, RT-PCR it and then did real time.
The control was the same RNA cloned into an expression vector. The cloned RNA was transcribed and the RNA quantified. Then RT-PCR and real time were dun.
Why go throw all this treble?
Because in this way you can eliminate the sum of errors from the efficiency of the RT-PCR and of the real time.

Hi,

Can you tell me which expression vector you have used for cloning the RNA. Mine is a single stranded RNA and hence how would I go about cloning this???
My virus is from stool samples and hence all the samples that will be used for the RT-PCR step to make the cDNA will be weighed, that is all the samples are weighed and then extracted and then RT-PCR will be carried out, I know this would not eliminate the problems from the RT-PCR step.
So would be happy if you could tell me which plasmid you have used for the cloning process.


thekid.

I would say you have to convert your RNA in cDNA, clone this cDNA into a vector, do an in vitro transcription (IVT), quantify your IVT RNA, reverse transcribe your IVT RNA back to cDNA and do a calibration curve. The problem: you don't know if the reverse transcription efficiency of your IVT RNA is the same as in your samples.

tea-test on Mar 30 2009, 04:53 PM said:

Yes, that's what we did.

One problem point is better then having a lot of them.

Hi I am also getting the same problem with plasmid standard preparation. I don't know if there is something wrong with my computation or the steps.
My goal is to develop a working standard for copy number work. Please see my preliminary result in my website. Please feel free to leave a comment.
http://mponscientic.blogspot.com/2010/05/c...timisation.html