I am using species-specific PCR primers targeted to the 16S rDNA gene. I am cloning the amplified fragment into a plasmid to be used to make the standard curve in quantitative PCR. Should I clone the entire 16S rDNA gene into the plasmid or is cloning the amplified product of the 16S rDNA gene sufficient?
As someone who has done this before, I'd recommend that you clone the entire 16S region. Why? Just in case you need to use a different set of primers for your qPCR. This will give you a lot more flexibility in your experimental design.
Good luck!
Normally the full length 18S rDNA is about 1500 to 2000 bp. It is a bit large to be amplified by PCR and cloned into the vector. For the qPCR product, it is normally less than 100 bp, it is too small to be ligated into the vector. Therefore, selection of about 1000 bp fragment, which contains the qPCR target sequence should be ideally for this purpose.
cloning the amplified product is sufficient
Indeed just incorporating the sequence you use to amplify in qPCR is actually more reprrsentative in order to titrate for your standard curve so actually more accurate
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