plasmids to quantify copies of virus - (Nov/20/2006 )

Hello everyone. I'm doing real time to detect a porcine virus. Now I want to quantify its copies to do a standard curve and a quantitative real time PCR. I started working with infected cell lines but I'm having problems. I'm thinking about expressing my target gene in a plasmid, transfect to E.coli colonies and try to quantfy from them but I am not sure if this is a good way to do it because in every article I read, they quantify infecting cell lines.
Some idea about this?? If it's viable could someone tell me the first steps I should do?? I have never worked with plasmids.

I would like to clarify some thought with you...

1. How can you express your target gene in a plasmid before you transfect into E.coli?
2. What do you mean by "quantifying infecting cell line"?
3. How is your method diferent from other article?

thank you

Hello:

I was misunderstood. I want to include my gene into a plasmid but before that, I amplify it with a conventional RT-PCR. When I have my gene into the plasmid, I would like to transfect the E.coli. I have to remark that I have never done it so maybe this is not the correct way to do it.
My aim is to have bacterias with the plasmid that has my gene, so I can (aproximately) quantify how many copies of my gene I have. (more or less I would do one plasmid=one bacteria)

When I talk about infected cell lines, I mean that usually, people on articles describe how they use a cell line where the virus grows and infect it with a known TCID50 and from that, they do a standard curve to use in their Real-Time PCR assays.

My method would be different because I wouldn't use cells to quantify the virus, instead of that, I would use plasmids into bacterias to quantify the virus.

Hope I cleared your doubts. (I'm not sure if this experiment is viable)
Thanks!!

Dear debokuki,

Your first post said that you are doing detection on a porcine virus. So I think absolute quantification may suit your purpose.
Absoulute quantifiatin meaning that you transtect your palsmid into bacteria --> growth the bacteria --> harvest the plasmid using plasmid extraction kit --> quantify the plasmid --> convert the spectrophotometer reading into molecular copy number --> and this is your quantitation stock.

From the stock (with known amount) --> serial dilution --> run real-time PCR --> and you will get standard cuve.