Bio rad cfx manager sample volume problem serial#
Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates.
We find that the estimated PCR efficiency varies significantly across different instruments. Dilutions of the ProNex DNA QC gDNA Standard in ProNex DNA QC Dilution Buffer can be stored at 4C for up to 1 week. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10 μl volume of analyte transferred, respectively. DNA QC Primer-Probe-IPC Mix, Bio-Rad CFX96 is light-sensitive and must be stored in the dark. 4 Specify a sample volume of 20 l and edit the protocol parameters to match those shown below. 3 On the Protocol tab of the software, click Edit Selected to open the Protocol Editor.
2 From the Express Load drop-down menu, select CFX2StepAmp. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. 1 In the CFX Manager software, click File > New > Experiment.
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates.
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