Hello,
I am doing DNA-SIP for 13C labeling soil samples lately and I checked the SCI papers which have done DNA-SIP work.
Generally, after ultracentrifugation, we do density gradient fractionation and measure with refractometer, then, we retrieve DNA from different fractions. Then, we do qPCR check of DNA from different fractions and and also from 12C control and compare the qPCR result. At first, I thought we need to get two peaks for 13C labled sample, light fraction and heavy fraction, respectively. For 12C, it should have only one peak in light fraction only.
However, I found that there are so many papers which got only peak in heavy fraction of qPCR check after ultra-centrifugation and fractionation.
But, some papers have two peaks in light and heavy fractions.
So, I would like to know if it is acceptable if we got only one peak in heavy fraction for qPCR check.
I am looking forward to your kindly help.
Thank you.
Best regards
Lynn
Hi Tin,
It depends on your sample. I'm assuming as you are labeling soil samples your DNA will be a mixed community (rather than a single species). With a mixed community it is common that you will see just one peak, spread over a range of fractions due to the different G+C content of the different bacteria in the sample.
Over time as labeling increases you should see an increase in DNA in the heavier factions. It is the increase in the heavier fractions over time you should be looking out for rather than the peak.
With a mixed community depending on the proportion of active members consuming the labelled substrate in the community your peak may not change much between labelled and unlabelled samples but over time you should see an increase in DNA in the heavy fractions.
Hope this helps,
Kathryn
Dear Kathryn, Kathryn Wigley
Thank you so much for your answer. It helps me a lot to think clearly about DNA-SIP result interpretation by qPCR.
best regards
Lynn
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