I am using three biological replicates. Their DNA concentrations measured by Qubit were different. I diluted all samples using an online calculator to ensure each sample had a concentration of 2.5 ng/μL. I used 2 μL of each sample in the qPCR, resulting in 5 ng/ul of DNA per well.
After performing qPCR, I noticed a slight difference in the Ct values of the biological replicates. I wondered why there was this difference. When I rechecked the DNA concentrations of my diluted biological replicates, I found slight variations i-e: 2.36 ng/μL, 1.98 ng/μL, 2.4 ng/μL, etc. This explained the observed differences in the Ct values during qPCR.
My question is: how can I ensure equal DNA concentrations in all samples?
Measure the dna concentration well into the working range of the spectrometer....errors are large at low OD and high OD values.
In making dilutions use large volumes of dna.....the errors involved in pipetting low volumes ( less than 5ul) are very large.
Dilute the dna so that you use 5ul or even 10ul volumes added to the pcr reaction as once again the pipetting errors are greatest at small volumes. For instance pipetting 1ul could easily have an error of 10% using routine and seldom calibrated pipettes.
clean and calibrate your pipettes regularly
Sumaira Shabbir
Make a master DNA stock, dilute it to the desired concentration, then aliquot it equally for each replicate. To guarantee consistency, repeat the measurement with Qubit and make any necessary adjustments. For qPCR, use calibrated pipettes and follow a standardised procedure.
You expect to see variation between *biological* replicates. That's why you have to include a "housekeeping/steady state/reference" gene to be able to compare between replicates. Now, you would hope for minimal to no variation within a set of *technical* replicates of the same biological sample. That will tell you if you have errors in pipetting.
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