Pipetting Academy: Best Pipetting Practices for qPCR 

Sensitivity of Quantitative Polymerase Chain Reaction (qPCR) based assays is one of the most important reasons for their success and abundant use in scientific laboratories – particularly with clinical diagnostics and biopharmaceuticals. It is frequently used for gene expression analysis by quantitating changes in gene expression from biological and environmental samples. However, factors like pipetting technique can influence qPCR assay results. 

Commonly Asked Questions  

• How can pipetting affect pPCR results?  - Because PCR Master Mix contains proteins, improper pipetting can lead to the creation of bubbles in the sample, which can skew the results of the experiment. 
• Which pipetting technique is best when using PCR Master Mix?  - According to ISO guidelines, it is recommended to pre-wet the pipette tip 3 to 5 times and use the reverse pipetting technique.
• Is pipette calibration important when using PCR Master Mix?  - There is a propensity for error when dealing with viscous materials such as PCR Master Mix. Therefore, it is important to eliminate as many risk factors as possible. Ensuring your pipette is regularly maintained and calibrated is an effective way to keep results accurate and reproducible. 
• Should I use a multichannel pipette for preparation of qPCR assays?  - Using a multichannel pipette for qPCR assays ensures equal amounts of PCR Master Mix will be evenly and quickly distributed into several wells at once. This is a time-saving factor especially when working with a 96 or 384 well plate. Reproducibility can be increased in experiments further by using an electronic pipette and multi-dispensing mode.