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qPCR , eDNA and standard curve

qPCR standard curve calc. eDNA

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#1 Garth Owen Watson

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Posted 07 March 2016 - 08:10 PM

I wish to use qPCR to detect a specific species of fish from eDNA in the water of aquaria tanks. Since eDNA is fragmented and therefore of differing sizes I would think it not suitable for use as a standard curve. Could i use PCR product instead?



#2 Trof

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Posted 08 March 2016 - 10:36 AM

You should clone your PCR product into a plasmid and transform a bacteria to get more plasmid, that can be isolated in high concentrations, measured and diluted with similar diluent as your real sample is (in human gDNA, plasmids are linearized and mixed with rRNA to simulate complexity of genomic DNA, you may not need that with your eDNA). This is how standards are commonly prepared.

 

PCR product can be used, when made fresh, if you need it just few times, but it's not that easy to quantify precisely and in time the ends will start to degrade (plasmid  is more stable) and also, you can't very well simulate the real template.


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#3 Garth Owen Watson

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Posted 08 March 2016 - 01:40 PM

I have since seen a recent paper use/order synthetic oligonucleotides (100 bases) then serial diluted for use as qPCR standard. This would be simpler since mine is a "one off". Are there obvious issues with this idea?


Edited by Garth Owen Watson, 08 March 2016 - 07:20 PM.


#4 Trof

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Posted 14 March 2016 - 02:54 PM

You have a PCR product smaller than 100 bp? The oligos, unless modified will have the same degradation problems as normal DNA, only benefit from PCR product is purity (and maybe longer overhangs, but I can't magine that on 100 bp oligo).

Putting PCR product into ready to use TA vector is very simple and if you have lab with wide scale of methods in use, you are likely to have all needed, from bacteria/vectors, incubators and kits for isolation. Of course if you have no microbiology stuff, then it is difficult.

Depends if it's for a single-time experiment or you want to do it in long term. In the first case I would go with PCR product, as I did in past, otherwise I would take the efford or pay someone to make a proper standard, stable and in large quantities.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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