Friday, December 6, 2013

Tips for Transferring Small Volumes in PCR (or Other) Experiments

This article is for those who are learning how to do PCR. Other articles that describe PCR are in the "Further Reading" section after the text. 

PCR stands for Polymerase Chain Reaction, a technique in biomedical research that is used to make copies of a certain length of DNA. 

PCR, reverse-transcription PCR (RT-PCR), and quantiative PCR (qPCR; qRT-PCR) involve handling sub-microliter volumes of liquids that need to be mixed.  One major source of experimental error is when people re-use one pipette tip for aliquot-ing small volumes of the same reagent, thinking that as long as they avoid contamination with the other reagents their experiment will be fine.  What they don’t realize is that the volume increases with each use of a micropipette tip. Thus, they will see trends in their replicates that should not be there, or the readings between replicates will be very sporadic.  The optimal way to do it is to use a new tip every time.  Also, to minimize user-introduced trends in the data, they should not aliquot a reagent into all replicates for treatment group A, and then to treatment group B.  There is no way to completely get rid of user imprecision, so one way around this is to aliquot sample A1, then B1; A2, then B2; A3, then B3, etc. (See Figure 1). These suggestions would apply to other assays that require distribution of small volumes from the same source. 

Figure 1

Further Reading

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