How can i make standard curve from PCR product

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qaiseruaf
qaiseruaf's picture
How can i make standard curve from PCR product

dear fellows,

i want to quantify my 16s r RNA gene using primer 338f/518r for bacterial community and 18s rRNA using primer NS1/Fung for fungal community in different soil samples.
i am going to use qPCR first time. i want to make standard curve from my PCR product to calculate number of copies.
can someone help me out how can i proceed for it to get good results.
thanks

qais

Ivan Delgado
Ivan Delgado's picture
 

 
Hi qais,
To run standard curves all you need to do is purify high quality DNA from your organism of interest. For example to prepare the standard curve for your bacterial community you need to isolate bacterial DNA from a typical sample and quantify it. The same is true for your fungal community. Now if you are trying to quantify multiple bacteria/fungi, then it gets a little trickier because every time to collect a specimen it will contain multiple DNAs and these may not amplify in the same way as your standard. One way to get around this would be to synthesize a piece of DNA that takes into account all the DNA differences you expect in your community. The drawback to this is that while you will be able to quantify total bacterial/fungal DNA in your sample, you will not know if the DNA came from one, multiple, or even the correct species. 
Trying to summarize: an absolute quantitation qPCR experiment requires the use of a standard curve of known DNA quantity. You need to identify what this DNA is (depends highly on what you are trying to quantify). Once you do that, you purify the DNA and quantify it, Then you prepare 5 dilutions (typically 10-fold dilutions) and run all five as separate qPCR assays. That is your standard curve. To quantify unknown samples you just interpolate the results you obtain from your unknown samples into the standard curve values. 
Hope this helps

qaiseruaf
qaiseruaf's picture
Dear Ivan,

Dear Ivan,

Thanks a lot dear friend for your kind and sincere suggestions regarding construction of standard curve for real time PCR.
i get your points, hope everything will go nicely.
have a good time.

Qais

kjerstilea
kjerstilea's picture
So the curve is based on the

So the curve is based on the initial, known concentration of the DNA - not the amount amplified in the PCR cycle? It is developed by observing when the known starting amount of template DNA is sufficiently amplified to cross the threshold of detectability Ct?