qPCR-growth curve - (Mar/21/2006 )

Hi all!

When I do qPCR of an axenic protist culture and evaluating the growth curve and in addition compare the date from the qPCR with microscopic counts, .. how do I take into account the sample volume, elution volume and dilution volume after my ICycler generated the Starting Quantity by the plasmid standard curve method?!

I hope you can help me!

Thanks Sil

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*sigh* ... another arithmetically challenged biologist (only joking ). You just have to normalise all of your data to units of 'per ml of culture'.

Your ICycler protocol probably gives you the copy number per reaction, or per ml of reaction volume (depending on how you entered the calibrator amounts - check that you know what your 'Starting Quantity' units are!). You know what volume of template (extracted DNA?) went into each reaction, so you can easily calculate the copy number per ul of extract ( C ). Presumably you also know what volume (V , ml) of the original culture you extracted, and the elution volume (E , ml) used, so V/E is your concentration factor (during the extraction). Divide C by this factor to get the copy number per ul of culture, then multiply by 1000 to get copy number per ml of culture.
Of course, this assumes 100% extraction efficiency...

If you have lots of these to do you should set up a spreadsheet (i.e. Excel) to do the calculations automatically as you enter your data. Check carefully that it works as expected using dummy data...!

D.

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Hi Del,

Thanks for your help, but I already found out the right equation:

Result (copies/ml)= (Result (copies/µl) x Elution volume (µl))/ Sample volume (ml)


But thanks anyway, Sil

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