Pipetting Error In Real Time Pcr
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Reverse Pipetting
that comparing them would be meaningless. But luckily, there's no need to worry—your trusty, precision micropipettes take care of that for you. Or do they? Precision instruments they may be, but the accuracy of your micropipettes depends on you. You need to maintain your pipettes, practice good technique, and have an understanding of how they work. Only once you have mastered those aspects can you claim to have anything like precision instruments at your disposal. Here's how: Know How Your Pipette Works 1. You Use an Air Displacement Pipette, Most Likely An air displacement pipette works a bit like a syringe, except that there is an air-filled cushion between the piston and the sample. The air cushion prevents the piston from coming into contact with the solutions. Keeping the solution and the pipette barrel apart is good, but it also puts some limitations on the pipette. Temperature and pressure affects the volume of the air cushion, which affects pipetting accuracy. Also, volatile solvents can evaporate into the air cushion, which leads to an inaccurate and lower dispensed volume than what is displayed on the pipette. The barrel of air displacement pipettes is also vulnerable to contamination by the p
on PCR amplification efficiencies See all blogAuthor:Jan HellemansAugust 07, 2014 Bioazelle has always validated quantitative PCR assays by combining specificity analysis (targeted sequencing or microfluidic electrophoresis) with an assessment of the PCR amplification efficiency from the slope of a standard curve or errors in pipetting technique dilution series, in accordance with theMIQE guidelines.Having wet-lab validated more than 100,000 assays [see how to pipette small volumes tech note 6262 for materials and methods; assays commercialized as PrimePCR assays by Bio-Rad] with an average efficiency of 99% and
Common Pipetting Errors
more than 98% of the assays with an efficiency of at least 90% [Figure 1] we were quite satisfied with the observed performance. However, recently we started to see more assays failing to meet our http://bitesizebio.com/344/17-ways-to-stop-pipetting-errors-ruining-your-experiments/ quality criterion of acceptable PCR efficiency within the 90-110% range. Nothing that would raise an eyebrow for a handful of assays – some assays are simply not good enough – but worrisome when seeing large numbers deviate. Not only did we observe a drop in efficiency but also a concurrent increase in the y-intercept of the standard curve. Once again this could indicate inferior assay performance, but it was suspicious https://www.qbaseplus.com/knowledge/blog/impact-pipetting-errors-amplification-efficiencies when observed as a trend across thousands of assays (we have a peak wet lab validation capacity of over 2000 assays per week).One of the potential causes for such a deviating trend is pipetting errors. However, imprecise pipetting would have been detected much earlier based on the coefficients of determination (r2) of the standard curves. Inaccurate pipetting might explain the observed drop in average amplification efficiency over the long run if it occurred in between our pipette calibrations or if the inaccuracy fell within the tolerated range of pipetting inaccuracy. To assess the impact of inaccurate pipetting on amplification efficiency determination using dilution series we performed a mathematical simulation study. This analysis indicates that every percentage of inaccurate pipetting (of the template in a 7-point 10-fold dilution series) (e.g. 9.9 µl instead of 10 µl)results in an efficiency drop of approximately 0.5%. This may seem small, but for a tolerance on pipetting accuracy of 10% this would increase the fraction of failed assays (efficiency below 90%) by 10-fold (from 1.6% to over 16%).Although the quality and reliability of qPCR, at least when performing relative quantification, does not depend too much on pipetting accuracy (precision remains important though), the assessment of amplification efficiencies from standard curves does require highly
the form of qPCR. As reproducibility is essential to genetic research it is imperative that scientists know the fundamentals of micro-volume pipetting. Forward and Reverse Pipetting: This discussion http://blog.biosearchtech.com/TheBiosearchTechBlog/bid/43104/Pipetting-for-qPCR is limited to the use of manual pipettors. Electronic pipettors are capable of other pipetting techniques such as dispensing, sequential dispensing and diluting which are not discussed here. Forward pipetting is used for aqueous solutions such as water, buffers, diluted saline, diluted acid or base. Appropriate aqueous solutions may also contain low concentrations of proteins or detergents. This technique is appropriate for milliliter and microliter volumes. how to In forward pipetting, aspiration involves compression of the key to the first stop followed by the slow release of the key, creating a vacuum within the barrel and aspirating the solution volume desired. Expulsion involves dispensing the solution by pressing the key down to and beyond the first stop to "blow out" the entirety of the aspirated volume. Reverse pipetting is used for viscous solutions, how to pipette solutions with high vapor pressure or extremely small microliter volumes. In reverse pipetting, aspiration involves compression of the key to the second stop followed by the slow release of the key, creating a vacuum within the barrel and the aspiration of a volume greater than that selected. Expulsion involves pressing the key down to the first stop only, thus dispensing only the desired volume. Pipetting micro-volumes: Pipette with smooth and deliberate action. Hold the pipette vertically at all times. This is best accomplished by using your index finger to dispense and aspirate instead of your thumb. Immerse the pipette tip only slightly to avoid coating the outside of the tip with excess liquid that may be inadvertently transferred during dispensing. Pipette the initial volume directly to the bottom of the receiving container while lifting the pipette upward slowly so as not to introduce bubbles to the dispensed solution. Add additional volumes to the initial volume using the same technique. Tips to improve accuracy: Pre-wet the tip. When pipetting greater than 10 microliters, it is good practice to do an initial aspiration and expulsion to decrease the amount of volume that will "stick" to the i