Construct DNA plasmid for QPCR - (Jun/06/2005 )

I'm trying to construct DNA plasmid for getting the standard curve for QPCR. My template is viral RNA (retrovirus). I cannot find the control plasmid from commerical sources as it's rare species. How could I do that? I'm thinking of the possible procedures like these below:

1. PCR Amplification of viral RNA
2. PCR gel clean from gel
3. Cloning the amplified DNA into the cloning vector (e.g. TOPO)
4. Use mini-prep to get the plasmid and do sequencing to confirm the identity of insert again
5. Measure the DNA or plasmid concentration

However, I do not know how to measure the plasmid concentration at last. The ideal situation will be that I could get the exact number of copy of plasmid in the prepared prep. How can I do that?

And I haven't tried making standard on my own before. What else I should pay attention to? Or are there any other more direct ways of having the standard curve on viral RNA/DNA - instead of plasmid standard?

Sonia

Hi Sonia,

Simply, you could just use PCR product as qPCR template to generate standard curve. It's much easier than using plasmid, at least avoiding cloning step and saving a lot of time.

Ruyang

I'm trying to construct DNA plasmid for getting the standard curve for QPCR. My template is viral RNA (retrovirus). I cannot find the control plasmid from commerical sources as it's rare species. How could I do that? I'm thinking of the possible procedures like these below:

1. PCR Amplification of viral RNA
2. PCR gel clean from gel
3. Cloning the amplified DNA into the cloning vector (e.g. TOPO)
4. Use mini-prep to get the plasmid and do sequencing to confirm the identity of insert again
5. Measure the DNA or plasmid concentration

However, I do not know how to measure the plasmid concentration at last. The ideal situation will be that I could get the exact number of copy of plasmid in the prepared prep. How can I do that?

And I haven't tried making standard on my own before. What else I should pay attention to? Or are there any other more direct ways of having the standard curve on viral RNA/DNA - instead of plasmid standard?

Sonia

Sonia -

In my experience, it doesn't matter exactly how much you are adding when you are generating your curve (i.e. specific ug of target DNA) but that you are very careful to do dilutions somewhere near the range of what you expect to get when you are running your assay

You could always make a plasmid yourself with the know sequence of your virus. Simply PCR clone the reagion and clone it into a topo cloning vector. Next grow up the plasmid and quantitiate, I have used the one-step RT-PCR reagents and used in vitro transcribed RNA and a target for my standard curve. In this case, I fell it better suits judging the initial RNA copy number.

Dear Sonia,

Using DNA and RNA as quantitative standard make alot of different.
Quantification of viral RNA using DNA as standard is not valid.
However, since retrovirus has both RNA and DNA stage in a cell, I think is depend on what you are trying to do?
Are you trying to determine the number of DNA intermediate or latent infaction of retrovirus. Or you are studying how many virus progeny being produced?


Best regards