I need to build a standard curve for qPCR of archaeal amoA using Sybr Green technology. I've seen that several papers use the primers designed by Tourna et al. (2008), and in turn, that paper refers to one by Treusch et al. (2005). The last author reports a cloned sequence (amoA-like seq) into fosmid 54d9. Can anyone tell me how to get this or an alternative DNA seq to work with?
Hi Maria,
The reference is not too old...get in touch with the author to get either the clone or the DNA for your analysis...or else clone this gene youself in a vector to make a std curve for analysis..
Hi Maria,
Have you amplified the product. If you have, you can clone it and it will become your internal standard. As you will get concentration of that plasmid and turn it into copy numbers
Hi María,
I think it is advisable to clone a PCR product yourself. You'll never run out of amoA gene copies if you keep transformants and grow them every now and then using common microbiology techniques. Plasmids with inserts are really stable in most conditions and provide excellent results in qPCR standard curves.
Some researchers do construct standard curves directly from PCR products but I do not like this procedure very much since quantification of the copy number in this standard samples can be rather tricky. Linearized fragments can be easily degraded and changes in the amount of intact fragments (copies) may occur, thus overestimating your samples.
Suerte
Lluis
Luis, I do plan to clone the fragment into a vector. The question is, would it be easy to amplify archaeal amoA directly from an agricultural soil as Ag Muhamad suggests (I've done so for bacterial amoA and it worked fine)? should I use any kind of soil or those preferred by archaea (acidic, low fertility, etc.)? That's why I am trying to get the "type srain" to build the standard and reduce the work towards qPCR of AOA
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